Herv-k antigens, antibodies, and methods

ABSTRACT

Methods and compositions for cancer diagnostics and therapy are provided. More particular, methods and compositions for detecting, preventing, and treating HERV-K +  cancers are provided. One example of a method may involve a method for preventing or inhibiting cancer cell proliferation by administering to a subject a cancer cell proliferation blocking or reducing amount of a HERV-K env protein binding antibody.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 11/752,235 and claims priority to U.S. patentapplication Ser. No. 11/752,235 filed on May 22, 2007 and U.S.Provisional Application Ser. No. 60/747,850 filed on May 22, 2006, theentirety of which is incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This disclosure was developed at least in part using funding from theDepartment of Defense, Grant No. DAMD1700-1-0123 and the Susan G. KomenBreast Cancer Foundation (BCTR0402892). The U.S. government may havecertain rights in the invention.

SEQUENCE LISTING

This disclosure includes a sequence listing submitted as a text filepursuant to 37 C.F.R. § 1.52(e)(v) named sequence listing.txt, createdon Sep. 4, 2007, with a size of 4,866 bytes, which is incorporatedherein by reference. The attached sequence descriptions and SequenceListing comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R. §§1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984). The symbols and format used for nucleotide andamino acid sequence data comply with the rules set forth in 37 C.F.R.§1.822.

BACKGROUND

Human endogenous retroviruses (HERVs) and elements containing longterminal repeat-like sequences may comprise up to 8% of the humangenome. HERVs entered the human genome after fortuitous germ lineintegration of exogenous retroviruses and were subsequently fixed in thegeneral population. They may have been preserved to ensure genomeplasticity and this can provide the host with new functions, such asprotection from exogenous viruses and fusiogenic activity (e.g.,membrane fusion, exocytosis, or endocytosis). HERVs contain over 200distinct groups and subgroups. The accumulation of mutations has led toa loss of infectivity of HERVs, and in general they are largelynoninfectious retroviral remnants. However, open reading frames (ORFs)have been observed for ERV3, HERV-E 4-1, and HERV-K, but theirsignificance is unknown.

The most biologically active HERVs are members of the HERV-K superfamilywhich is characterized by the presence of primer binding sites forlysine tRNA. Only HERV-K appears to have the full complement of openreading frames typical of replication competent mammalian retroviruses.The K family contains a central open reading frame (cORF) and iscomparable to HIV-1 Rev protein. HERV-K was originally identified by itshomology to the mouse mammary tumor virus (MMTV), and istranscriptionally active in several human cancer tissues, includingbreast cancer tissues, as well as tumor cell lines, such as the humanbreast cancer cell line T47D and the teratocarcinoma cell line GH.

HERV-K env mRNA is frequently expressed in human breast cancer andHERV-E mRNA is expressed in prostate cancer. Additionally, mRNA frommultiple HERV families is transcribed only in ovarian cancer cell linesand tissues. For example, the expression of HERV-K env mRNA was greaterin ovarian epithelial tumors than it was in normal ovarian tissues(N=254).

Breast cancer is a significant health problem for women in the UnitedStates and throughout the world. Although advances have been made indetection and treatment of the disease, breast cancer remains the secondleading cause of cancer-related deaths in women, affecting more than180,000 women in the United States each year. For women in NorthAmerica, the life-time odds of getting breast cancer are now one ineight.

No vaccine or other universally successful method for the prevention ortreatment of breast cancer is currently available. Management of thedisease currently relies on a combination of early diagnosis (throughroutine screening procedures) and aggressive treatment, which mayinclude one or more of a variety of treatments such as surgery,radiotherapy, chemotherapy, and hormone therapy. The course of treatmentfor a particular breast cancer is often selected based on a variety ofprognostic parameters, including an analysis of specific-tumor markers.See, e.g., Porter-Jordan & Lippman, Breast Cancer 8:73-100, 1994.However, the use of established markers often leads to a result that isdifficult to interpret, and the high mortality observed in breast cancerpatients indicates that improvements are needed in the treatment,diagnosis, and prevention of the disease.

Ovarian cancer is another leading cause of cancer deaths among women andhas the highest mortality of any of the gynecologic cancers. Symptomsusually do not become apparent until the tumor compresses or invadesadjacent structures, or ascites develops, or metastases becomeclinically evident. As a result, two thirds of women with ovarian cancerhave advanced (Stage III or IV) disease at the time of diagnosis.

Potential screening tests for ovarian cancer include the bimanual pelvicexamination, the Papanicolaou (Pap) smear, tumor markers, and ultrasoundimaging. The pelvic examination, which can detect a variety ofgynecologic disorders, is of unknown sensitivity in detecting ovariancancer. Although pelvic examinations can occasionally detect ovariancancer, small, early-stage ovarian tumors are often not detected bypalpation due to the deep anatomic location of the ovary. Thus, ovariancancers detected by pelvic examination are generally advanced andassociated with poor survival. The pelvic examination may also producefalse positives when benign adnexal masses (e.g., functional cysts) arefound. The Pap smear may occasionally reveal malignant ovarian cells,but it is not considered to be a valid screening test for ovariancarcinoma. Ultrasound imaging has also been evaluated as a screeningtest for ovarian cancer, since it is able to estimate ovarian size,detect masses as small as 1 cm, and distinguish solid lesions fromcysts.

Serum tumor markers are often elevated in women with ovarian cancer.Examples of these markers include carcinoembryonic antigen, ovariancystadenocarcinoma antigen, lipid-associated sialic acid, NB/70K, TAG72.3, CAI 15-3, and CA-125, respectively. Evidence is limited on whethertumor markers become elevated early enough in the natural history ofoccult ovarian cancer to provide adequate sensitivity for screening, andtumor markers may have limited specificity.

Tumor-associated antigens recognized by the immune system are a veryattractive target for human cancer diagnostics and therapy. However, fewimmunotherapy approaches have been used for the treatment and preventionof cancers. One problem limiting the success of cancer vaccines is thatthe immune system generally does not recognize cancer cells as beingforeign, which is a requirement for initiating an immune response.Cancer immunotherapy, however, is limited due in part to the limitednumber of tumor-associated antigens identified to date.

SUMMARY

The present disclosure, according to specific example embodiments,generally relates to HERV-K⁺ cancers.

The present disclosure is based in part on the observation that HERV-Ksurface env protein has antigenic and immunogenic properties. HERV-K envprotein may not be expressed, or expressed at low-levels, in normal orbenign tissues. This may lead to the production of T cells that are notautoreactive. Thus, the present disclosure uses HERV-K env proteins asunrecognized tumor associated antigens in HERV-K⁺ cancers.

The involvement of the HERV-K env protein in host immune functions thusmakes it of potential use in HERV-K⁺ cancer diagnosis and treatment.Accordingly, the present disclosure provides methods of preventing orinhibiting HERV-K⁺ cancers, such as breast and ovarian cancers, cellproliferation, and diagnosing or staging cancers. The present disclosurealso provides HERV-K env protein-specific antibodies; and relatedmethods of using these materials to detect the presence of HERV-K envproteins or nucleic acids.

The present disclosure also advantageously provides for screening assaysand kits, such as methods of screening for compounds that inhibit orprevent HERV-K⁺ cancer proliferation

The present disclosure also provides HERV-K⁺ cancer specific antigenthat may be used for, among other things, in vitro expansion of HERV-K⁺cancer-specific CD8⁺ cytotoxic T lymphocytes (CTLs) for autologoustransfer. Such antigens also may be used to generate anti-HERV-Kantibodies and to detect the presence of anti-HERV-K antibodies inHERV-K⁺ cancer patients.

The present disclosure also provides autologous dendritic cells (DCs)pulsed with HERV-K env protein, peptides, and cRNAs. Such DCs enableautologous professional antigen presenting cells to process and presentone or more HERV-K epitopes in association with host human leukocyteantigen (HLA) molecules. HERV-K env antigens are capable of breakingtumor patient immune tolerance, and the IVS cells subsequently generatedare capable of killing HERV-K⁺ target cells.

The present disclosure also provides HERV-K env protein for use as,among other things, a diagnostic marker for endometrioid, serous, mixedmullerian tumors (MMT), poorly differentiated, and transitionalcarcinoma. Expression of HERV-K env SU protein was significantlyincreased in low malignant potential serous tumors and endometrioidtumors, compared with normal ovaries.

The present disclosure also provides antibodies against HERV-K env SUprotein, HERV-K gag protein, HERV-K spliced envelope protein, HERV-Esurface protein, or ERV3 env protein.

The features and advantages of the present disclosure will be readilyapparent to those skilled in the art upon a reading of the descriptionof the embodiments that follows.

FIGURES

Some specific example embodiments of the disclosure may be understood byreferring, in part, to the following description and the accompanyingdrawings.

FIG. 1A is an illustration of HERV-K env protein expression in tumorepithelial cells obtained from a patient with infiltrating ductalcarcinoma. Detection of HERV-K env protein expression in tumorepithelial cells obtained from a patient with infiltrating ductalcarcinoma. Serial breast tissue sections obtained from a breast cancerpatient were assessed by immunohistochemistry using antibody specificagainst HERV-K env protein. The expression of HERV-K env protein wasdetected only in tumor epithelial cells, including ductal carcinoma insitu (DCIS) and invasive ductal carcinoma (IDC), and not in uninvolvednormal epithelial cells (C).

FIG. 1B is an illustration of examples of immunostaining withanti-HERV-K env antibody in a multiple-tissue microarray. Case #1,normal mammary lobule from a 43-year-old female; case #4, normal mammarylobule from a 50-year-old female; case #16, mammary gland tissue from a61-year-old female; case #8, IDC (grade II) from a 45-year-old female;case #17, IDC (grade II; 49 year-old female); Case #11, intraductalcarcinoma (grade II) from a 52-year-old female.

FIG. 1C is a graph of HERV-K env protein expression in two microarraysof 126 breast tissue samples. “1” indicates low expression, “2”indicates intermediate expression, and “3” indicates strong expressionof HERV-K env protein. The levels of expression were associated withtissue type (cancer, benign, and normal) (P<0.001; Fisher's exact test).

FIG. 1D is a summary of HERV-K env protein expression in three arrays of182 breast tissue samples (0 indicates no expression; 1 indicates lowexpression; 2 indicates intermediate expression; 3 indicates strongexpression). The expression levels were associated with tissue type(cancer, benign, and normal) (P<0.001; Chi-square test). Two of theseven benign breast biopsies (ductal epithelial hyperplasia) were weaklyHERV-K positive (low expression). More than 50% of breast cancerbiopsies had intermediate or strongly positive staining for HERV-K.

FIG. 2A is a graph of ELISA detection of antibodies against anti-HERV-Kenv surface protein (K-SU), gag protein (K-gag), and spliced env protein(K-spliced) in the sera from cancer patients and normal female controlsubjects. Sera were diluted 1:200. The ELISA plate was read at awavelength of 405 nm, with a cutoff of ≦0.5 absorbance units.

FIG. 2B is a graph of ELISA detection of IgG antibody against HERV-K envsurface protein in plasma from cancer patients and normal female controlsubjects. Plasma was diluted 1:100. Only IgG antibodies from plasmabinding HERV-K env surface protein were detected by this assay. Thelevel of anti-HERV-K IgG antibodies in plasma from cancer patients(N=14) was higher (P<0.001) than the level in plasma from normal controlsubjects (N=12).

FIG. 3A are graphs of phenotyping of immature and mature human DCs byflow cytometry. Immature DCs were exposed to TNF-α overnight formaturation, with or without prior pulsing with HERV-K proteins. MatureDCs not stained with antibody were used as negative control cells andDCs stained with single antibody were used as compensation controls(data not shown). The percentage of CD86⁺/CD83⁺ DCs, CD209⁺/CD83⁺ DCs,and CD209⁺/CD86⁺ DCs obtained from immature DCs (immature), mature DCswithout (mature) or with HERV-K (HERV-K pulsed) prior pulsing with areshown.

FIG. 3B are graphs of determination of surface expression of HERV-K envprotein on HERV-K-pulsed mature DCs. The percentage of surfaceexpression of HERV-K on DCs was determined by flow cytometry usinganti-RGS mAb (anti-RGS; as a positive control) or anti-HERV-K specificantibody (anti-HERV-K).

FIG. 3C is an illustration of the expression of HERV-K env protein onhuman breast cancer cells by flow cytometry and fluorescence microscopy.The cells were permeabilized (permeabilized) for detection ofcytoplasmic expression, or not (non permeabilized) for detection ofsurface expression. Cells stained with anti-IgG-FITC were used asnegative controls (data not shown). The same cells used for flowcytometry were subjected to fluorescence microscopy (micrographs areshown in insets). Surface and cytoplasmic expression of HERV-K in MCF-7,but not in MCF-10AT cells is shown.

FIG. 4A is an illustration of T-cell proliferation results from PBMCsobtained from control subjects (N=7). The T cell proliferation wascompared between PBMCs and CD3+ T cells obtained from the same donors.Results are shown for_PBMCs or CD3+ T cells without protein stimulation(‘Cells only’); cells stimulated with HERV-K env protein (‘HERV-K’);cells stimulated with HERV-E env protein (‘HERV-E’); cells stimulatedwith the superantigen Staphylococcus enterotoxin A (‘SEA’). The data arepresented as corrected mean counts per minute per 1×10⁵ PBMCs or CD3⁺ Tcells. All assays were done in triplicate.

FIG. 4B is a graph of proliferation results from IVS cells. Each donorwas tested for proliferation of PBMCs stimulated with unpulsed DCs andIVS cells stimulated with HERV-K pulsed DCs. The proliferation indexdata obtained from IVS/PBMC were compared between the cancer patientsand control subjects. The proliferation was higher in IVS obtained fromcancer patients than in IVS obtained from control subjects (P=0.025).

FIG. 4C is a graph of antigen-specific granzyme B producing cells, asassessed by ELISPOT analysis. Granzyme B spots obtained from IVS orPBMCs were compared between cancer patients and control subjects. Thegranzyme B spots were higher in IVS obtained from cancer patients thanin IVS obtained from control subjects (P=0.003).

FIG. 4D is a graph of HERV-K-specific lysis of target cells.Determination of HERV-K-specific lysis of target cells. Cytotoxic T cellassay of 3-week IVS cells obtained from two cancer patients. Targetcells were K562 cells used to assess natural killer activity, autologousDCs pulsed with HERV-K env protein (DC+K) or with control protein(DC+Mock), MCF-7 breast cancer cells, or autologous B-LCL cells pulsedwith HERV-K (B-LCL+K) or with control protein (B-LCL+Mock). The ratio ofeffector cells to target cells was 20:1.

FIG. 5A is a graph of cytokine secretion by IVS cells obtained frombreast cancer patients and normal control subjects showing the meanvalues of IL-2 secretion from cancer patients (82 pg/ml) and normalsubjects (22 pg/ml). The mean values of IL-2 secretion from cancerpatients (82 pg/ml) and normal subjects (22 pg/ml) are shown. IL-2secretion was higher (P=0.029) in IVS cells from cancer patients(Cancer; N=13) than in IVS from normal female donors (Normal; N=17).

FIG. 5B is a graph of cytokine secretion by IVS cells obtained frombreast cancer patients and normal control subjects showing the meanvalues of IFN-γ secretion from cancer patients (535 pg/ml) and normalsubjects (144 pg/ml). IFN-γ secretion is significantly higher (P=0.028)in IVS cells from breast cancer patients than in IVS from normal femaledonors.

FIG. 5C is a graph of cytokine secretion by IVS cells obtained frombreast cancer patients and normal control subjects showing the meanvalues of IL-6 secretion from breast cancer patients (7,448 pg/ml) andnormal subjects (1,306 pg/ml). IL-6 secretion was higher (P=0.038) inIVS cells from breast cancer patients (Cancer) than in IVS from normalfemale donors (Normal).

FIG. 5D is a graph of cytokine secretion by IVS cells obtained frombreast cancer patients and normal control subjects showing the meanvalues of IL-8 secretion from breast cancer patients (16,360 pg/ml) andnormal subjects (8,793 pg/ml). IL-8 secretion was higher (P=0.028) inIVS cells from breast cancer patients (Cancer) than in IVS from normalfemale donors (Normal).

FIG. 6A are graphs of intracellular TNF-α, IL-2 and IFN-γ production byPBMCs and HERV-K-specific IVS cells, as assessed by intracellularcytokine staining. PBMCs, or IVS cells obtained by stimulating PBMCsfrom the same donor with HERV-K pulsed DCs, were either not activated asnegative controls (Unactivated), nonspecifically activated with aleukocyte activation cocktail as positive controls (Non-specific), oractivated with HERV-K plus brefeldin A (HERV-K-activated). IncreasedHERV-K-activated cytokine production was observed in the IVS cells only(CD3⁺ T cells).

FIG. 6B are graphs of intracellular TNF-α, IL-2 and IFN-γ production byPBMCs and HERV-K-specific IVS cells, as assessed by intracellularcytokine staining. PBMCs, or IVS cells obtained by stimulating PBMCsfrom the same donor with HERV-K pulsed DCs, were either not activated asnegative controls (Unactivated), nonspecifically activated with aleukocyte activation cocktail as positive controls (Non-specific), oractivated with HERV-K plus brefeldin A (HERV-K-activated). IncreasedHERV-K-activated cytokine production was observed in the IVS cells only(CD3⁺ T cells).

FIG. 6C is a graph of the results of a cytokine bead array, used todetermine HERV-K-specific cytokine production after 1 week IVS. IL-2secretion was significantly elevated in 1 week IVS from BC patients, incomparison to PBMC obtained from BC patients (N=17; p=0.0159; Student'st-test). In contrast, no significant change was observed in 1 week IVSfrom normal controls, in comparison to PBMC obtained from normalcontrols (N=15).

FIG. 6D is a graph of the results of a cytokine bead array, used todetermine HERV-K-specific cytokine production after 1 week IVS. Similarto IL-2, IFN-γ secretion was significantly elevated in 1 week IVS fromBC patients, in comparison to PBMC obtained from BC patients (N=17;p=0.0034; Student's t-test). As with IL-2 normal control subjects, nosignificant difference in IFN-γ secretion was observed between 1 weekIVS and PBMC obtained from normal controls (N=15). IFN-γ secretion wassignificantly lower (p=0.0263) in PBMC obtained from BC patients than inPBMC from normal controls, which suggests that BC patients may beimmunosuppressed.

FIG. 7A is an illustration of expression of HERV env RNAs in ovariancell lines and tissues by RT-PCR for ERV3, HERV-E, HERV-K type 1, HERV-Ktype 2, and β-actin primers in OVCAR3 ovarian cancer cells, NOE 114normal ovarian epithelial cells, and SKOV3 ovarian cancer cell. Celllines: From left to right, each set of lanes for a given amplified generepresents the RT-PCR expression pattern using ERV3, HERV-E, HERV-K typeI (HERV-K (1)), HERV-K type 2 (HERV-K (2)), and β-actin primers inOVCAR3 ovarian cancer cells (lane 1), NOE 114 normal ovarian epithelialcells (lane 2) and SKOV3 ovarian cancer cell (lane 3). 4. The final lanein each set is a no-template control (lane 4).

FIG. 7B are illustrations of expression of HERV env RNAs in ovarian celllines and tissues by RT-PCR for ERV3, HERV-E, HERV-K type 1, and HERV-Ktype 2 env mRNA in matched tumor/normal tissues. Expression of variousHERV env mRNAs was evaluated in two cancer tissues (lanes 1 and 3) withtheir matched uninvolved normal ovarian tissues (lanes 2 and 4) obtainedfrom the same patients. The final lane in each set is a no-templatecontrol (lane 5).

FIG. 7C is an illustration of expression of HERV env RNAs in ovariancell lines and tissues by RT-PCR for spliced HERV-K transcripts. Lanes1-7: each lane represents a different ovarian cancer specimen. The finallane is a no-template control (lane 8). Full-length (2) and Full-length(1) represent unspliced full-length HERV-K type-2 and type-1transcripts, respectively.

FIG. 7D is a graph quantifying HERV-K env mRNA in various ovariantissues. The amount of HERV-K in unknown samples was quantitated usingcycle threshold (C_(T)) values of HERV-K env mRNA obtained from eachspecimen by real-time RT-PCR, normalized on the basis of the C_(T) ofHomo sapiens ribosomal protein S9. The ratio of HERV-K mRNA C_(T) inovarian tumor tissues to the average C_(T) in normal ovarian controltissues was calculated. The boxplot gives the upper extreme value (theline on the top), upper quartile (the top of box), median (the whiteband inside the box), mean (the ‘X’ symbol), lower quartile (the bottomof box), and lower extreme (the line on the bottom) for each box.Boxplots not only show the location and spread of data but indicateskewness as well. For this case, the bulb-peak distance has a smalleraverage value and variation than the it-peak distance. From left toright: 1. Benign epithelial tumor; N=6; 2. Epithelial tumor with lowpotential, N=5; 3. Mixed epithelial tumor, N=11; 4. Normal ovariancontrols, N=19; 5. Placenta (as a control), N=13; 6. Uninvolved ovariantissues, N=12; 7. Epithelial tumor with metastasis, N=46; 8. Epithelialtumor without metastasis, N=121; and 9. Sex cord and stromal tumor(including germ cell tumor), N=21. HERV-K env expression wassignificantly greater (lower C_(T)) in tissues from epithelial tumorwithout metastasis (p=0.012), and epithelial tumor with metastasis(p=0.058), relative to expression in normal and benign ovarian controltissues.

FIG. 8A are illustrations of surface expression of HERV-K env protein onovarian cancer cells. Surface and cytoplasmic expression of HERV-K envprotein was detected in ovarian cancer cell lines (OVCA 420 and DOV13),but not in normal ovarian epithelial cells (T29 and T80) withoutpermeabilization (No-perm) and permeabilized with 0.1% Triton X-100(Perm). Surface and cytoplasmic expression of HERV-K env protein wasdetected in DOV13 cells by FACS analysis. Cells stained with FITC-IgG Abserved as a negative control.

FIG. 8B is a graph of expression of HERV proteins in various ovariantissues.

FIG. 8C are illustrations of samples exhibiting positive immunostainingfor HERV-K from TMA1 microarray: a. Normal ovarian tissues (score “0”;40×). b. Clear cell carcinoma (score “1”). c. Serous papillarycystadenocarcinoma (score “2”). d. Serous papillary adenocarcinoma(score “3”).

FIG. 8D are illustrations of samples exhibiting positive immunostainingfor HERV-K from TMA2 microarray: a. Mucinous cyst. b. Mucinous LMP (lowmalignant potential). c. LG (Low-grade) endometrioid. d. HG (High-grade)endometrioid.

FIG. 8E are illustrations of are illustrations of samples exhibitingpositive immunostaining for HERV-K from TMA2 microarray: e. Serous LMP.f. LG Serous. g. HG Serous. h. Clear cell carcinoma.

FIG. 9A is a graph of an expression profile of HERV-K env SU proteinexpression in serous papillary adenocarcinoma of various grades (I, IIand III). Percentage of “no expression” progressively decreased fromlower to higher grades, whereas percentage of “strong expression”progressively increased from lower to higher grades.

FIG. 9B is a graph of an analysis of ovarian cancer progression withtissue microarrays: 1. Normal ovary. 2. Mucinous cyst. 3. Mucinous tumorof low malignant potential. 4. Serous tumor of low malignant potential.5. Low-grade serous carcinoma. 6. Low-grade endometrial carcinoma. 7.High-grade serous carcinoma. 8. High-grade endometrial carcinoma. 9.Clear cell carcinoma. Low malignant potential and low-grade tumorsshowed higher levels of expression compared to normal ovarian surfaceepithelial cells (Kruskall Wallis analysis p<0.001). High-grade tumorsshowed great variability in protein expression with a median expressionslightly lower compared to normal ovaries.

FIG. 10A is a graph of binding affinity and specificity of anti-HERV-Ksera from 20 patients with ovarian cancer. An ELISA plate was coatedwith HERV env fusion proteins including HERV-K envelope surface protein(K-SU), HERV-E surface protein (E-SU), HERV-K gag protein (K-gag),HERV-K plus (a HERV-K spliced env product), and ERV3 env protein. Seraobtained from 20 patients with ovarian cancer were tested. The ELSIAplate was read at a wavelength of 405 nm. The cutoff value is 0.5 for ODat 405 nm.

FIG. 10B is a graph of binding affinity and specificity of anti-HERV-Ksera from 20 normal female controls. An ELISA plate was coated with HERVenv fusion proteins including HERV-K envelope surface protein (K-SU),HERV-E surface protein (E-SU), HERV-K gag protein (K-gag), HERV-K plus(a HERV-K spliced env product), and ERV3 env protein. Sera obtained from20 normal female controls were tested. The ELSIA plate was read at awavelength of 405 nm. The cutoff value is 0.5 for OD at 405 nm.

FIG. 11A is a graph of the binding affinity and specificities ofanti-HERV-K monoclonal antibodies from an ELISA analysis of bindingaffinity and specificities of the positive clones derived fromanti-HERV-K hybridoma cells. The ELISA plate was coated with HERV-K orHERV-E env fusion proteins (10 μg per ml, 100 μl per well). The mediaobtained from several positive clones were diluted from 1:50 to1:109,350. The ELISA plate was read at a wavelength of 405 nm.

FIG. 11B is a graph of the binding affinity and specificities ofanti-HERV-K monoclonal antibodies from an ELISA analysis of bindingaffinity and specificities of the positive clones derived fromanti-HERV-K hybridoma cells. Various concentrations of HERV-K envprotein were coated on ELISA plates, and the medium obtained from twohybridoma clones (4D11 and 6H5) and two negative controls were dilutedfrom 1:20, to 1:2,000. These positive clones, but not two negativecontrols, reacted with only HERV-K env fusion protein.

FIG. 11C is an image illustrating binding of mAb clones 4D1 or 6H5 toHERV-K env SU fusion protein, confirmed by Western blot. Antibodiesagainst HERV-K env protein in sera obtained from patients with BC (BC1,BC2, and BC3), but not in sera from a normal donor (CON1) weredemonstrated by Western blot.

FIG. 11D is a graph illustrating titration of antibodies against HERV-Kenv SU protein in sera from BC patients, accomplished by ELISA. The seraobtained from normal female donors were used as controls. The frequencyof antibodies detected in patients with BC (N=31) and normal donors(N=20) is shown. Anti-HERV-K SU antibody titers were significantlyhigher in BC patients than in normal donors.

FIG. 12 is a graph showing that an anti-HERV-K antibody inhibitsproliferation of breast (MCF-7) and ovarian (DOV13) cancer cell lines,but not normal breast (MCF-10A) or ovarian (T80) cell lines. Humanepithelial cells were treated with anti-HERV-K antibody (5693) orpreimmune sera (CS) on day 1 and day 4. Proliferation of cells wasmeasured by the MTT assay. Values represent the mean of six replicatewells at days 1, 4, and 7 of culture.

FIG. 13A is a graph showing splice donor (SD) (SEQ ID. NO:1) and spliceacceptor (SA) sites (SEQ ID. NO:2) for HERV-K subgenomic transcriptsfrom human breast cancer tissue. Samples #165U2 and #165U4 are locatedat bp numbers 1076 and 6433, respectively, according to the type 2HERV-K, HML-2.HOM sequence.

FIG. 13B is a graph showing SD (SEQ ID. NO:3) and SA (SEQ ID. NO:4)sites for HERV-K subgenomic transcripts from human breast cancertissues. Samples #165U3 and 165U5 are located at bp numbers 876 and5997, respectively, according to the type 1 HERV-K, HERV-K102 sequence.

FIG. 13C is a graph showing SD (SEQ ID. NO:5) and SA (SEQ ID. NO:6)sites for HERV-K subgenomic transcripts from human breast cancertissues. Samples #178U11 and 178U15 are located at bp numbers 928 and6399, respectively, according to the type 1 HERV-K, HERV-K102 sequence.

FIG. 13D is a graph showing SD (SEQ ID. NO:7) and SA (SEQ ID. NO:8)sites for HERV-K subgenomic transcripts from hormone-treated T47D cellslocated at bp numbers 883 and 6222, respectively, according to the type1 HERV-K102 sequence.

FIG. 13E is a graph showing SD (SEQ ID. NO:9) and SA (SEQ ID. NO: 10)sites for HERV-K subgenomic transcripts from hormone-treated MCF-7 orMDA-MB-231 cells are located at bp numbers 2078 and 7599, respectively,according to the type 1 HERV-K (II) sequence.

FIG. 14A is a graph showing the anti-tumor effect of HERV-K env proteinantigen in mice. Mice were inoculated with B6DK cells (5×10⁶ cells) onday 0 and randomly divided into groups and treated with bone-marrow DCpulsed with nothing, HERV-K env protein (DC+K pro); control protein(DC+control pro), HERV-K cRNA (DC+KcRNA); or with control cRNA(DC+control cRNA) on day 3, day 10, and day 17 post-injection. Tumorswere monitored twice per week and tumor sizes were compared between eachgroup.

FIG. 14B is a graph showing is a graph showing the anti-tumor effect ofHERV-K env protein antigen in mice. Mice were inoculated with B6DK cells(5×10⁶ cells) on day 0 and randomly divided into groups and treated withbone-marrow DC pulsed with HERV-K cRNA (DC+KcRNA), HERV-K env derivedpeptide for surface protein (Kp201) or transmembrane protein (Kp640),DNA methyl transferase I (p1028; as positive control), and nothing onday 7, day 14, and day 21 post-injection. DC pulsed with HERV-K envprotein, cRNA, or even peptides elicit a strong antitumor response toB6DK (*p<0.05) compared with mice treated with DC only.

FIG. 15 are DNA and protein sequences of anti HERV-K scFv. (A) DNAsequences of anti HERV-K scFV (SEQ ID. NO:11); bold sequence at 5′ endis restriction site for SfiI, and sequence at 3′ end is restriction sitefor NotI. Bold sequence in the middle is linker sequence. Sequencebetween SfiI site and linker is heavy chain scFv sequence, and sequencebetween linker and NotI is light chain scFv sequence. (B) Amino acidsequences of anti HERV-K scFV (SEQ ID. NO:12).

FIG. 16A illustrates HERV-K expression in BC cells. Surface (none-perm)and cytoplasmic (perm) expression of HERV-K env protein was detected onMDA-MB-231 BC cells by staining unpermeabilized (Non-perm) andpermeabilized (Penn) cells, respectively.

FIG. 16B illustrates HERV-K expression in BC cells. HERV-K env proteinexpression was detected on MCF-7 BC cells by immunofluorescence using alaser scanning confocal microscope and anti-HERV-K monoclonal antibody.

FIG. 16C illustrates HERV-K expression in BC cells. HERV-K env proteinexpression was not detected on benign MCF-10A breast cells. Observationswere made under a laser scanning confocal microscope. Observations weremade from top to bottom of the cells using z-sectioning.

FIG. 16D illustrates HERV-K expression in BC cells. The percentage ofpositive surface expression of HERV-K env protein was greater on MCF-7cells (55%) than on MCF-10A cells (5%), by FACS analysis.

FIG. 16E illustrates HERV-K expression in various cell lines. Theexpression of HERV-K env protein in various breast cell lines and anovarian cancer cell line was detected by Western blot assay using 6H5mAb against HERV-K env surface protein. β-actin was used as control.(top). FIG. 16E (bottom panel) shows that anti-HERV-K antibodies weredetected in sera obtained from breast cancer patients.

FIG. 16F shows that anti-HERV-K antigen antibodies were detectable inbreast cancer sera. Serial dilutions of patients were tested in ELISAassays for antibody activity against HERV-K, Np9, and Rec recombinantproteins.

FIG. 17A shows detection of HERV-K-specific T cell proliferation.HERV-K-specific T-cell proliferation in BC patient PBMC compared tonormal donor PBMC, as determined by ³H-thymidine incorporation in PBMCor IVS cells. Each donor was tested for stimulation of proliferation byDC pulsed with nothing (cell only), HERV-K SU protein (DC+K pro), HERV-KSU cRNA (DC+KRNA), and HPV16E6 protein (DC+E6pro). Proliferation wasdetermined in IVS cells incubated one time with DCs pulsed with HERV-Kenv surface protein. T cell proliferation was increased in 3 of 4 IVSobtained from cancer patients compared to 0 of 4 IVS cells obtained fromnormal donors. No difference was observed in PBMC proliferation when BCpatients and normal donors were compared. HPV16 E6 protein produced froma pQE30 expression vector or HERV-K env surface cRNA were used ascontrols.

FIG. 17B shows detection of HERV-K-specific T cell proliferation.HERV-K-specific T-cell proliferation in BC patient PBMC compared tonormal donor PBMC, as determined by ³H-thymidine incorporation in PBMCor IVS cells. Each donor was tested for stimulation of proliferation byDC pulsed with nothing (cell only), HERV-K SU protein (DC+K pro), HERV-KSU cRNA (DC+KRNA), and HPV16E6 protein (DC+E6pro). A similar T cellproliferation result was obtained for IVS cells incubated one time withDC pulsed with HERV-K env surface cRNA produced by in vitrotranscription. HPV16 E6 protein produced by pQE30 vector or HERV-K envsurface cRNA was used as control.

FIG. 17C shows detection of HERV-K-specific T cell proliferation.HERV-K-specific T-cell proliferation in BC patient PBMC compared tonormal donor PBMC, as determined by ³H-thymidine incorporation in PBMCor IVS cells. Each donor was tested for stimulation of proliferation byDC pulsed with nothing (cell only), HERV-K SU protein (DC+K pro), HERV-KSU cRNA (DC+KRNA), and HPV16E6 protein (DC+E6pro). The T cellproliferation index was obtained from each donor's 1-week IVS, comparedwith PBMC stimulation by HERV-K-pulsed DC. The proliferation index washigher in BC patients (5.632±1.812; N=16) than in normal donors(1.388±0.4735; N=18; P=0.023; Student's t-test).

FIG. 18A illustrates detection of HERV-K-specific CD8⁺ T response.Antigen-specific GrB- or IFN-γ producing cells, as assessed by ELISPOTanalysis. ELISPOT was performed on unstimulated PBMC from a BC patient(BC) and a normal control subject (NL) or after 1-week IVS withHERV-K-pulsed DC. DC pulsed HPV16 E6 protein served as the control. Agreater number of GrB- or IFN-γ spots were detected in IVS cellsproduced from DC pulsed with HERV-K env surface protein obtained from BCpatients than from normal donors.

FIG. 18B illustrates detection of HERV-K-specific CD8⁺ T response. GrBspots obtained from IVS cells were compared between cancer patients andcontrol subjects after stimulation with HERV-K-pulsed DC. The GrB spotnumbers were higher in IVS cells obtained from cancer patients(233.9±46.26 N=13) than in IVS cells obtained from control subjects(74.73±16.67 N=16; P=0.0016; t tests).

FIG. 18C illustrates detection of HERV-K-specific CD8⁺ T response. A CTLassay was performed after 1-week IVS from four BC patients and fourhealthy female donors. Autologous DC pulsed with HERV-K env protein(DC+Kpro) or cRNA (DC+KRNA), as well as DC pulsed with HPV16E6 protein(DC+E6pro) were used as target cells. Unlabeled K562 cells were used tocorrect for nonspecific lysis. The ratio of effector cells to targetcells was 100:1, 50:1, 25:1, and 12.5:1.

FIG. 19A shows comparative cytoxicity of 6H5 mAb towards breast celllines (left) or ovarian cell lines (right). Cells were treated withmedium containing different concentrations of 6H5 for 72 h, the cellswere then stained with crystal violet and read at 595 nm. Cells withoutantibody treatment were used for controls. The percent inhibition ofcell growth is shown. IC₅₀: 50% inhibitory concentration.

FIG. 19B illustrates that anti-HERV-K antibody is able to induce MCF-7cells to undergo apoptosis, compared with cells without Ab treatment(control). The right bottom panel represents cells that are Annexin V⁺and 7AAD⁻ (17% in early apoptosis) and the right top panel representscells that are Annexin V⁺ and 7AAD⁺ (23% in late apoptosis).

FIG. 19C summarizes the results of the apoptosis studies in breast celllines. The top figures show a summary of the effect of dose of 6H5 oninduction of breast cells to undergo apoptosis, compared with cellswithout Ab treatment (control). The bottom figures show a summary of theeffect of various mAb clones on induction of apoptosis in breast cells.

FIG. 20A illustrates that adoptive T cell therapy in mice inhibitedbreast tumor growth.

FIG. 20B illustrates tumor formation in mice innoculated with MCF-7cells on day 0 and treated with saline or 6H5 on days 4, 6, and 8(arrows; 200 ug per mice). Mice treated with saline were used ascontrol.

FIG. 21 illustrates western blot of various ovarian cancer cell linesusing 6H5 mAb to detect expression of HERV-K env protein.

FIG. 22 illustrates detection of surface (Non-perm) and cytoplasmic(Perm) expression of HERV-K env protein in ovarian cancer cells byconfocal microscopy using 6H5 mAb. rGel was delivered into DOV13 cellsby 6H5, and was detected by anti-rGel Ab.

FIG. 23 illustrates detection of surface (Non-perm) and cytoplasmic(Perm) expression of HERV-K env protein in breast cell lines by confocalmicroscopy using 6H5 mAb. rGel was delivered into cells by 6H5, and wasdetected by anti-rGel Ab.

FIG. 24 illustrates detection of surface (Non-perm) and cytoplasmic(Perm) expression of HERV-K env protein in breast cell lines by confocalmicroscopy using 6H5 mAb. rGel was delivered into cells by 6H5, and wasdetected by anti-rGel Ab.

FIG. 25 illustrates quantitation of surface expression of HERV-K envprotein in ovarian or breast cell lines by dry ELISA using 6H5 mAb.Murine IgG was used as a negative control.

FIG. 26 illustrates quantitation of surface expression of HERV-K envprotein in ovarian or breast cell lines by FACS using 6H5 mAb. MurineIgG was used as a negative control.

FIG. 27 illustrates that 6H5 mAb is able to induce ovarian cancer cellsto undergo apoptosis, compared to cells without Ab treatment (control;top panels). The bottom panels represent cells that are Annexin V⁺ and7AAD⁻ (right bottom, in early apoptosis) and the right top panelrepresents cells that are Annexin V⁺ and 7AAD⁺ (right top, in lateapoptosis).

FIG. 28 shows a summary of 6H5 induction of ovarian cells to undergoapoptosis, compared to cells without Ab treatment (control.) Blue bar isearly apoptosis and red bar is late apoptosis.

FIG. 29 illustrates Coomasie Blue staining of 6H5 mAb (lane 1) and6H5-rGel conjugate (Lane 2) in non-reducing gel.

FIG. 30 illustrates comparative cytotoxicity of 6H5-rGel, 6H5, and rGelalone toward MCF-10A and MDA MB453 cells. Cells were treated with mediumcontaining different concentrations of 6H5-rGel, 6H5, and rGel alone for72 h and stained with crystal violet and read at 595 nm. The percentageof growth inhibition relative to control cell growth is shown. IC50 foreach cell line is listed in the table.

FIG. 31 illustrates comparative cytotoxicity of 6H5-rGel, 6H5, and rGelalone towards ovarian T29 and ovarian cancer DOV13 cells. Cells weretreated with medium containing different concentrations of 6H5-rGel,6H5, and rGel alone for 72 h and stained with crystal violet and read at595 nm. The percentage of growth inhibition relative to control cellgrowth is shown. IC50 for each cell line is listed in the table.

FIG. 32A illustrates expression of HERV env RNAs in melanoma cell linesand tissues. HERV-K expression in normal melanocytes (HEMn-LP; lane 1and HEMn-DP; Lane 2), in human malignant melanoma cells (SK-MEL-28cells; lane 3 and SK-MEL-1; Lane 4), and in melanoma biopsies (lanes 5and 6) obtained from patients. Expression of both of types of HERV-K envmRNAs was detected in melanoma cancer cells and biopsies (Lanes 3 to 6).

FIG. 32B illustrates expression of HERV env RNAs in melanoma cell linesand tissues. Purified Np9/GST and Rec/GST recombinant fusion proteinswere detected by Coomassie blue staining.

FIG. 33 illustrates expression of HERV-K env protein in melanomatissues. A. The expression of HERV-K env protein in malignant melanomaCase No. 4 (score “3”); B. metastasis to lymph node of case No. 4 (score“3”); C. HERV-K expression in melanoma (score “2”); and D. expression inmelanoma metastasis to lymph node (score “1”).

FIG. 34A illustrates detection of anti-HERV antigen antibodies inmelanoma patient sera. Serial dilutions of patient sera were tested inELISA for antibody activity against HERV K, Np9, and Rec recombinantproteins.

FIG. 34B illustrates detection of anti-HERV antigen antibodies inmelanoma patient sera. An initial screen of sera from patients withdifferent cancer types found that melanoma patients have enhancedantibody reactivity against HERV antigens, especially Np9 and Rec.

FIG. 35 illustrates the results of a CTL assay on HERV-K-specific Tcells obtained after IVS with HERV-K Env protein or RNA. HERV-K specificT cells obtained from a melanoma patient (blue color) and one healthydonor (red) were used as effector cells, and autologous DC pulsed withHERV-K Env protein (DC+Kpro) or HERV-K RNA (DC+KRNA), DC pulsed withHPV16E6 protein (DC+E6 pro) or HPV16E6 RNA (DC+E6 RNA) were used astarget cells. K562 cells were added to neutralize nonspecific lysis. Theratio of effector cells to target cells was 50:1, 25:1, 12.5:1 and6.25:1.

FIG. 36 depicts the amino acid and nucleotide sequences of the 4D1 scFvits variable heavy and light chains and CDRs. (SEQ ID NOS. 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30).

FIG. 37 depicts the amino acid and nucleotide sequences of the 6H5 scFvgenerated, its variable heavy and light chains, and CDRs. (SEQ ID NOS.49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66).

FIG. 38 shows HERV-K protein as expressed in breast cancer cells anddetected by Western blot using 6H5 mAb.

FIG. 39 shows HERV-K env glycoprotein in breast cancer cells as detectedby Western blot using 6H5 mAb.

FIGS. 40 a, 40 b, 40 c and 40 d show the expression of HERV-K asdetected in lung carcinoma (A), colonic adenocarcinoma (B), gallbladdercarcinoma (C) and melanoma (D)

FIGS. 41 a, 41 b, 41 c, and 41 d show that HERV-K is not expressed innormal tissues including lung (A), heart (B), brain (C) and smallintestine (D) using 6H5 mAb in a tissue array with multiple tissuesincluding cancer and normal cells.

FIG. 42 shows that HERV-K is not expressed in normal tissues includinglymph node (E), thyroid (F), pancreases (G) and salivary glands (H)using 6H5 mAb in a tissue array with multiple tissues including cancerand normal cells.

FIG. 43 shows that HERV-K is not expressed in normal tissues includingendometrium (I), tongue (J), breast (K) and colon (L) using 6H5 mAb in atissue array with multiple tissues including cancer and normal cells.

FIG. 44 shows that HERV-K env protein is expressed in colon cancer (Aand B) and pancreas from cancer (C) and non-neoplastic (D) tissue by IHCusing 6H5 mAb.

FIG. 45 shows the number of molecules of HERV-K surface env protein invarious breast cells was quantified by QIFI assay using 6H5 mAb.

FIG. 46 shows the number of surface HERV-K env protein molecules inmelanoma cell lines was determined by flow cytometry using 6H5 mAb. TheMFI from each cell line was calculated according to the calibrationequation.

FIG. 47 shows that HERV-K env antigen was detected in several melanomacell lines including 888A2-Mel (top panel), 624-Mel (2nd panel) andA-375 (3rd panel) cells by dry cell ELISA and immunofluorescencestaining using 6H5 mAb. MCF-10AT breast cells 4th panel were used as anegative control.

FIG. 48 shows the expression profiles of HERV-K env protein on thesurface of various cancer cells or normal cells were evaluated andcompared by Q1F1 assay using 6H5 mAB.

FIG. 49 shows the ability of HERV-K recombinant protein to block bindingof anti-HERV-K antibodies to the cell surface was evaluated bypre-incubating the 6H5 with HERV-K recombinant protein (1 ug/10 ug 6H5).

FIG. 49 shows the ability of HERV-K recombinant protein to block bindingof anti-HERV-K antibodies to the cell surface was evaluated bypre-incubating the 6H5 with HERV-K recombinant protein (1 ug/10 ug 6H5).

FIG. 50 shows the cycling of HERV-K env protein between the cell surfaceand intracellular stores in breast cells. The percentage ofinternalization at 45 min was 39% for T47D cells, 57.92% for MCF-7, and64.52% for MDA-MB-231 cells, respectively.

FIG. 51 shows the net cellular uptake rates of anti-HERV-K antibodies.Surface quenching allows for distinction of surface and internalantibody fractions. Total cellular fluorescence was measured at eachtime point by flow cytometry and the internal and surface fractionsdetermined by surface quenching with an anti-Oregon green IgG.

FIG. 52 shows cell surface proteins were pulsed with biotin using anHS-SS-biotin reagent and chased at 37° C. At each time point (0, 5, 15,45, 90, and 180 minutes), cells were lysed, biotinylated proteins pulleddown with streptavidin resin and the pull down blotted for HERV-K.

FIG. 53 shows the results of MTS and cytotoxicity assays of cellstreated with anti-HERV-K mAbs. MCF-7 cells were treated with severalconcentrations of 6H5 or 6E11 mAb or mlgG on day 0, and cellproliferation was measured by MTS assay (OD 492 nm; left) orcytotoxicity assay (crystal violet staining; OD 600 nm; right) after 72hr.

FIG. 54 provides the results of cytotoxicity assays of cells treatedwith anti-HERV-K mAbs. There was no cytotoxicity of 6H5 toward MCF-10Anormal breast cells, in contrast to the significant cytotoxicity of 6H5toward MCF-7 and MDA-MB-231 breast cancer cells.

FIG. 55 shows the effect of antibody treatment on apoptosis of melanomacells. “untreated” represents cells not exposed to 6H5 or its scFv,“6H5-rGel” or “scFv” represent cells treated with 10 μg per ml of therespective antibodies. The effect of 6H5-rGel (6H5 conjugated to rGeltoxin) was not significantly different from the effect of its scFv onmelanoma cell apoptosis.

FIG. 56 shows the effect of 6H5 (red curve) or 6H5-rGel (blue curve) oninduction of apoptosis in breast cells, in comparison to the same cellsnot treated with 6H5 or 6H5-rGel (cells stained with anti-mouse IgG;gray color region).

FIG. 57 shows the expression of caspase 3, 8, and 9 was detected incancer cells treated with 6H5 (10 ug/ml) for 24 h by Western blot usingantibodies for caspase 3, 8, and 9.

FIG. 58 shows breast cells were treated with 6H5 or mlgG (10 ug/ml) for72 h and BrdU Incorporation was expressed as a % of control cells (cellstreated with mlgG).

FIG. 59 shows the cell cycle arrest in the G1/G0 phase (T47D) and Sphase (MCF-7, MDAMB231, and ZR75-1). Cells were induced with 6H5 mAb (10ug/ml) on days 1, 2, and 3.

FIG. 60 shows the results of the CDC assay of breast cancer cell lines:Breast cells were treated with 6E11 mAb (1 or 10 μg/ml) in media with1:5 to 1:30 dilutions of normal human sera complement. A greaterpercentage of negative P.I. staining is indicative of living cells.

FIG. 61 shows both 6H5 and 6E11 mAbs (10 μg/ml) induced MCF-7 cell deathby ADCC, using PBMCs from normal donors (top panel). Controls containedno mAb or no effector cells. The effector cell (E) to target cell (T)ratio was evaluated in the range of 25:1 to 6.25:1 (top left panel), andin the range of 100:1 to 25:1 (top right panel). The percentage of ADCClysis differed among individuals.

FIG. 62 provides the results of female SCID mice inoculatedsubcutaneously with MDA-MB-231 or MCF-7 (5×106 cells) on day 0 andtreated with mlgG, 6H5 or 6H5-rGel on days 4, 6, and 8 (arrows). Tumorsizes were measured twice per week, and average tumor volumes (L×W×D)for each group were compared

FIG. 63 shows the results of a TUNEL or Ki-67 assay was used fordetection of apoptosis or cell proliferation, respectively, inMDA-MB-231 human breast cancer tumors from SCID mouse xenografts. Micewere treated with 6H5 mAb for 1 or 2 weeks, and comparisons were madebetween the tumors of 6H5 mAb treated mice and control mice treated withsaline or mlgG (0).

FIG. 64 shows the results of MCF-7 cells treated with 6H5 or mlgG (10ug/ml) for 24 h, and 3 or 84 key genes involved in apoptosis, orprogrammed cell death were upregulated by 6H5 using human apoptosis PCRarrays. The fold changes in response to antibody treatment are shown.

TNFRSF25: tumor necrosis factor receptor superfamily, member 25

TNFSF8: tumor necrosis factor (ligand) superfamily, member 8

CIDEA: cell death-inducing DFFA-like effector a

FIG. 65 shows the detection of CIDEA in breast cancer cell lines ZR75-1and MDAMB231. Cells were treated with 6H5 mAb, and compared to the samecells treated with mlgG.

FIG. 66 shows the results of MCF-7 cells treated with 6H5 or mlgG (10ug/ml) for 24 h, and TWIST1 and MMP1, genes involved in invasion andmetastasis of cancer cells, were downregulated by 6H5 mAb. Data wereobtained using the Cancer Finder Pathway Superarray, and the foldchanges are shown in this graph.

TWIST1: Probably transcription factor

MMP1: collagenase-1

FIG. 67 shows the results of MCF-7 cells treated with 6H5 or mlgG (10ug/ml) for 24 h and the expression of 84 genes related to p53-mediatedsignal transduction was evaluated using a p53 Signaling Pathway PCRArray. Genes on the array include p53-related genes involved in theprocesses of apoptosis, the cell cycle, cell growth, proliferation, anddifferentiation, and DNA repair.

FIG. 68 shows humanized 4D1 scFv as generated in a human antibodyframework with murine CDRs as labeled (heavy chain is blue and lightchain is yellow). (SEQ ID NOS. 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49)

FIG. 69 shows humanized 6H5 scFv as generated in a human antibodyframework with murine CDRs as labeled (heavy chain is blue and lightchain is yellow) (SEQ ID NOS. 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84).

FIG. 70 shows GNP (white arrows) was detected by TEM in MDAMB231 cellsof tumors isolated from mice 24 hr post-i.v.-injection with 6H5-GNP.HERV viral particles (green arrows) were observed adjacent to tumorcells.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

While the present disclosure is susceptible to various modifications andalternative forms, specific example embodiments have been shown in thefigures and are herein described in more detail. It should beunderstood, however, that the description of specific exampleembodiments is not intended to limit the disclosure to the particularforms disclosed, but on the contrary, this disclosure is to cover allmodifications and equivalents as illustrated, in part, by the appendedclaims.

DESCRIPTION

Identification of unique cancer antigens enables the design of selectiveimmunotherapy for neoplastic diseases. The capacity to utilize adeterminant exclusively expressed by cancer cells, and which is devoidin normal tissues, ensures the targeting and elimination of theneoplastic cells while insulating the function of normal cells.

GENERAL DEFINITIONS

“human endogenous retrovirus” (HERV) is a retrovirus that is present inthe form of proviral DNA integrated into the genome of all normal cellsand is transmitted by Mendelian inheritance patterns. Such provirusesare products of rare infection and integration events of the retrovirusunder consideration into germ cells of the ancestors of the host. Mostendogenous retroviruses are transcriptionally silent or defective, butmay be activated under certain conditions. Expression of the HERV mayrange from transcription of selected viral genes to production ofcomplete viral particles, which may be infectious or non-infectious.Indeed, variants of HERV viruses may arise, which are capable of anexogenous viral replication cycle, although direct experimental evidencefor an exogenous life cycle is still missing. Thus, in some cases,endogenous retroviruses may also be present as exogenous retroviruses.These variants are included in the term HERV for the purposes of thedisclosure. In the context of the disclosure, human endogenousretrovirus includes proviral DNA corresponding to a full retroviruscomprising two LTRs, gag, pol, and env, and further includes remnants or“scars” of such a full retrovirus, which have arisen as a results ofdeletions in the retroviral DNA. Such remnants include fragments of thefull retrovirus, and have a minimal size of one LTR. Typically, theHERVs have at least one LTR, preferably two, and all or part of gag,pol, or env.

The term “isolated” means that the referenced material is removed fromthe environment in which it is normally found. Thus, an isolatedbiological material can be free of cellular components, i.e., componentsof the cells in which the material is found or produced. In the case ofnucleic acid molecules, an isolated nucleic acid includes a PCR product,an isolated mRNA, a cDNA, or a restriction fragment. In anotherembodiment, an isolated nucleic acid is preferably excised from thechromosome in which it may be found, and more preferably is no longerjoined to non-regulatory, non-coding regions, or to other genes, locatedupstream or downstream of the gene contained by the isolated nucleicacid molecule when found in the chromosome. In yet another embodiment,the isolated nucleic acid lacks one or more introns. Isolated nucleicacid molecules include sequences inserted into plasmids, cosmids,artificial chromosomes, and the like. Thus, in a specific embodiment, arecombinant nucleic acid is an isolated nucleic acid. An isolatedprotein may be associated with other proteins or nucleic acids, or both,with which it associates in the cell, or with cellular membranes if itis a membrane-associated protein. An isolated organelle, cell, or tissueis removed from the anatomical site in which it is found in an organism.An isolated material may be, but need not be, purified.

The term “purified” refers to material that has been isolated underconditions that reduce or eliminate the presence of unrelated materials,i.e., contaminants, including native materials from which the materialis obtained. For example, a purified protein is preferably substantiallyfree of other proteins or nucleic acids with which it is associated in acell; a purified nucleic acid molecule is preferably substantially freeof proteins or other unrelated nucleic acid molecules with which it canbe found within a cell. Purity can be evaluated by chromatography, gelelectrophoresis, immunoassay, composition analysis, biological assay,and other methods known in the art.

A “sample” refers to a biological material which can be tested for thepresence of HERV-K env protein or HERV-K env protein nucleic acids. Suchsamples can be obtained from subjects, such as humans and non-humananimals, and include tissue, especially mammary glands, ovaries,biopsies, blood, and blood products; plural effusions; cerebrospinalfluid (CSF); ascites fluid; and cell culture.

The term “non-human animals” includes, without limitation, laboratoryanimals such as mice, rats, rabbits, hamsters, guinea pigs, etc.;domestic animals such as dogs and cats; and, farm animals such as sheep,goats, pigs, horses, and cows.

The term “transformed cell” refers to a modified host cell thatexpresses a functional protein expressed from a vector encoding theprotein of interest. Any cell can be used, but preferred cells aremammalian cells.

The term “assay system” is one or more collections of such cells, e.g.,in a microwell plate or some other culture system. To permit evaluationof the effects of a test compound on the cells, the number of cells in asingle assay system is sufficient to express a detectable amount of theHERV-K env protein mRNA and protein expression. The methods of thedisclosure are suitable cells of the disclosure that are particularlysuitable for an assay system for test ligands that modulatetranscription and translation of the HERV-K env gene.

The terms “cancer” or “tumors” refers to group of cells that displayuncontrolled division. In a specific embodiment, the cancer is a HERV-K⁺cancer. In a specific embodiment, the cancer is breast cancer andparticularly infiltrating ductal and/or lobular carcinomas. In anotherspecific embodiment, the cancer is ovarian cancer. Ovarian cancer refersto any cancer in any of the three kinds of ovarian tissue cell types,which include germ cells, stromal cells, or epithelial cells. Themajority of epithelial tumor types are HERV positive. The term “cellproliferation” refers to the growth of a cell or group of cells.

The term “humanly acceptable” refers to compounds or antibodies that aremodified so as to be useful in treatment of human diseases or disorders.In a specific embodiment, antibodies (polyclonal or monoclonal) aremodified so that they are humanly acceptable. In one embodiment, thisrequires the antibodies to be humanized or primatized.

The use of italics generally indicates a nucleic acid molecule (e.g.,HERV-K env protein cDNA, gene, and the like); normal text generallyindicates the polypeptide or protein. Alternatively, whether a nucleicacid molecule or a protein is indicated, it can be determined by thecontent.

The term “amplification” of DNA refers to the use of polymerase chainreaction (PCR) to increase the concentration of a particular DNAsequence within a mixture of DNA sequences. For a description of PCR seeSaiki et al., Science, 239:487, 1988.

The term “nucleic acid molecule” refers to the phosphate ester form ofribonucleosides (RNA molecules) or deoxyribonucleosides (DNA molecules),or any phosphoester analogs, in either single stranded form, or adouble-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNAhelices are possible. The term nucleic acid molecule, and in particularDNA or RNA molecule, refers only to the primary and secondary structureof the molecule, and does not limit it to any particular tertiary forms.Thus, this term includes double-stranded DNA found, among other things,in linear (e.g., restriction fragments) or circular DNA molecules,plasmids, and chromosomes. In discussing the structure of particulardouble-stranded DNA molecules, sequences may be described according tothe normal convention of giving only the sequence in the 5′ to 3′direction along the nontranscribed strand of DNA (i.e., the strandhaving a sequence homologous to the mRNA). A “recombinant DNA molecule”is a DNA molecule that has undergone a molecular biologicalmanipulation.

The terms “polynucleotide” or “nucleotide sequence” is a series ofnucleotide bases (also called “nucleotides”) in DNA and RNA, and meansany chain of two or more nucleotides. A nucleotide sequence typicallycarries genetic information, including the information used by cellularmachinery to make proteins and enzymes. These terms include double orsingle stranded genomic and cDNA, RNA, any synthetic and geneticallymanipulated polynucleotide, and both sense and antisense polynucleotide.This includes single- and double-stranded molecules, i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases, for examplethiouracil, thio-guanine and fluoro-uracil.

The polynucleotides may be flanked by natural regulatory (expressioncontrol) sequences, or may be associated with heterologous sequences,including promoters, internal ribosome entry sites (IRES) and otherribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells can further be used for screening or other assays, as describedbelow.

Generally, a DNA sequence having instructions for a particular proteinor enzyme is “transcribed” into a corresponding sequence of RNA. The RNAsequence in turn is “translated” into the sequence of amino acids whichform the protein or enzyme. An “amino acid sequence” is any chain of twoor more amino acids. Each amino acid is represented in DNA or RNA by oneor more triplets of nucleotides. Each triplet forms a codon,corresponding to an amino acid. The genetic code has some redundancy,also called degeneracy, meaning that most amino acids have more than onecorresponding codon.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include regulatory DNA sequences, such as promoter sequences,which determine for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Anexpression product can be characterized as intracellular, extracellularor secreted. The term “intracellular” means something that is inside acell. The term “extracellular” means something that is outside a cell. Asubstance is “secreted” by a cell if it appears in significant measureoutside the cell, from somewhere on or inside the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence toa host cell, so that the host cell will express the introduced gene orsequence to produce a desired substance, typically a protein or enzymecoded by the introduced gene or sequence. The introduced gene orsequence may also be called a “cloned” or “foreign” gene or sequence,may include regulatory or control sequences, such as start, stop,promoter, signal, secretion, or other sequences used by a cell's geneticmachinery. The gene or sequence may include nonfunctional sequences orsequences with no known function. A host cell that receives andexpresses introduced DNA or RNA has been “transformed” and is a“transformant” or a “clone.” The DNA or RNA introduced to a host cellcan come from any source, including cells of the same genus or speciesas the host cell, or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. Vectors include plasmids, phages, viruses, etc.

A common type of vector is a “plasmid,” which generally is aself-contained molecule of double-stranded DNA, usually of bacterialorigin, that can readily accept additional (foreign) DNA and which canreadily introduced into a suitable host cell. A plasmid vector oftencontains coding DNA and promoter DNA and has one or more restrictionsites suitable for inserting foreign DNA. A large number of vectors,including plasmid and fungal vectors, have been described forreplication and/or expression in a variety of eukaryotic and prokaryotichosts. Non-limiting examples include pKK plasmids (Clontech), pUCplasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREPplasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New EnglandBiolabs, Beverly, Mass.), and many appropriate host cells, using methodsdisclosed or cited herein or otherwise known to those skilled in therelevant art. Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes.

A “cassette” refers to a DNA coding sequence or segment of DNA thatcodes for an expression product that can be inserted into a vector atdefined restriction sites. The cassette restriction sites are designedto ensure insertion of the cassette in the proper reading frame.Generally, foreign DNA is inserted at one or more restriction sites ofthe vector DNA, and then is carried by the vector into a host cell alongwith the transmissible vector DNA. A segment or sequence of DNA havinginserted or added DNA, such as an expression vector, can also be calleda “DNA construct.”

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells and Baculovirus vectors, and mammalian hostcells and vectors.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g. DNA, or any process, mechanism, or result of such achange. This includes gene mutations, in which the structure (e.g. DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g. protein or enzyme)expressed by a modified gene or DNA sequence. The term “variant” mayalso be used to indicate a modified or altered gene, DNA sequence,enzyme, cell, etc., i.e., any kind of mutant.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. See, Sambrook, Fritsch & Maniatis, Molecular Cloning: ALaboratory Manual, Second Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. The conditions of temperature and ionicstrength determine the “stringency” of the hybridization. Forpreliminary screening for homologous nucleic acids, low stringencyhybridization conditions, corresponding to a T_(m) (melting temperature)of 55° C., can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and noformamide; or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringencyhybridization conditions correspond to a higher T_(m), e.g., 40%formamide, with 5× or 6×SCC. High stringency hybridization conditionscorrespond to the highest T_(m), e.g., 50% formamide, 5× or 6×SCC. SCCis a 0.15M NaCl, 0.015M Na-citrate. Hybridization requires that the twonucleic acids contain complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of Tm forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived. See, Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. For hybridization with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity. See, Sambrook, Fritsch & Maniatis, Molecular Cloning: ALaboratory Manual, Second Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. A minimum length for a hybridizablenucleic acid is at least about 10 nucleotides; preferably at least about15 nucleotides; and more preferably the length is at least about 20nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C. In a specific embodiment, “highstringency” refers to hybridization and/or washing conditions at 68° C.in 0.2×SSC, at 42° C. in 50% formamide, 4×SSC, or under conditions thatafford levels of hybridization equivalent to those observed under eitherof these two conditions.

The term “oligonucleotide” refers to a nucleic acid, generally of atleast 10, preferably at least 15, and more preferably at least 20nucleotides, preferably no more than 100 nucleotides, that ishybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNAmolecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. In one embodiment, a labeled oligonucleotide can be used asa probe to detect the presence of a nucleic acid. In another embodiment,oligonucleotides (one or both of which may be labeled) can be used asPCR primers, either for cloning full length or a fragment of HERV-K Env,or to detect the presence of nucleic acids encoding HERV-K Env.Generally, oligonucleotides are prepared synthetically, preferably on anucleic acid synthesizer.

“Antibody-dependent cell-mediated cytotoxicity” and ADCC refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and subsequently cause lysisof the target cell. The primary cells for mediating ADCC, NK cells,express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII.

An “epitope,” as used herein is a portion of a polypeptide that isrecognized (i.e., specifically bound) by a B-cell and/or T-cell surfaceantigen receptor. Epitopes may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides derived from the nativepolypeptide for the ability to react with antigen-specific antiseraand/or T-cell lines or clones. An epitope of a polypeptide is a portionthat reacts with such antisera and/or T-cells at a level that is similarto the reactivity of the full length polypeptide (e.g., in an ELISAand/or T-cell reactivity assay). Such screens may generally be performedusing methods well known to those of ordinary skill in the art, such asthose described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. B-cell and T-cell epitopes may alsobe predicted via computer analysis. Polypeptides comprising an epitopeof a polypeptide that is preferentially expressed in a tumor tissue(with or without additional amino acid sequence) are within the scope ofthe present disclosure.

Cancers

As mentioned above, the term “cancer” refers to cells that displayuncontrolled proliferation or division. The degree to which a cancer hasspread beyond its original location is referred to as the “stage” of thecancer. Lower stages, such as stages I and II, are generally moreconfined to their site or region of origin than advanced stages (III andIV). See, e.g., The Merck Manual, 15^(th) Ed., Merck, Sharp, & DohmeResearch Laboratories (1987).

HERV-K⁺ cancers refer to cancers that are characterized by expression ofa HERV-K gene, or a polymorphism or sequence variant thereof, whichresults in an antigen derived from the HERV-K gene, or a polymorphism orsequence variant thereof. Examples of a HERV-K⁺ cancer include, but arenot limited to, breast cancers, ovarian cancers, teratocarcinomas, andmelanomas.

Breast cancers refer to a class of cancers that are associated withdevelopment in the breast of women and men. The most common type ofbreast cancer is invasive ductal carcinoma. It occurs most frequently inwomen in their 50's and appears to spread from the breast into the lymphnodes. The HERV-K env gene may be expressed in breast cancer cell lines,tumors, and tissues.

Ovarian cancer refers to a class of cancers that are associated withdevelopment in the ovaries of women. Carcinoma of the ovary is mostcommon in women over age 60. The most common type of ovarian cancer isepithelial ovarian carcinomas. The HERV-K env transcripts, as well astype 1 and type 2 HERV-K full length transcripts, may be detected inovarian cancer cell lines.

Polypeptides

The present disclosure describes polypeptides that encompass amino acidsequences encoded by a polynucleotide having a HERV-K env sequence, andvariants of such polypeptides. In specific embodiments, polypeptidesalso include polypeptides (and epitopes thereof) encoded by DNAsequences that hybridize to a HERV-K env sequence under stringentconditions, wherein the DNA sequences are at least 80% identical inoverall sequence and wherein RNA corresponding to the nucleotidesequence is expressed at a greater level in a cancer tissue than in thecorresponding normal tissue. Examples of such DNA sequences include, butare not limited to those shown in FIGS. 13A-E (SEQ ID. NO:1-SEQ ID.NO:10), and those listed in Table 5, and Table 6.

Cloning and Expression of HERV-K Env Protein

The present disclosure contemplates analysis and isolation any antigenicfragments of HERV-K env protein from any source, preferably human. Itfurther contemplates expression of functional or mutant HERV-K envprotein for evaluation, diagnosis, or therapy.

Conventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art may be employed in the use ofthis disclosure. Such techniques are explained fully in the literature.See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, Second Edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II(D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Antibodies

The present disclosure describes antibodies that bind HERV-K env proteinin cells and specifically in cancer cells such as HERV-K⁺ cancer cells,for example breast and ovarian cancers and melanoma. According to thedisclosure, HERV-K env polypeptides produced recombinantly or bychemical synthesis, and fragments or other derivatives, may be used asan immunogen to generate antibodies that recognize the HERV-K envpolypeptide or portions thereof. Such antibodies include, but are notlimited to, polyclonal, monoclonal, humanized, primatized, chimeric,single chain, Fab fragments, and a Fab expression library. An antibodythat is specific for human HERV-K env protein may recognize a wild-typeor mutant form of HERV-K env protein. In particular embodiments,antibodies are produced to, but not limited to, HERV-K env proteins, andvariants thereof. Specific examples of such antibodies include, but arenot limited to, antibodies that are capable of binding to HERV-K envsurface protein products from both types of HERVOK env regions, such as,HERV-K10 (HUMERVKA), HERV-K102 (AF164610), HERV-K103 (AF164611),HERV-K104 (AF164612), HERV-K107 (AF164613), HERV-K108 (AF164614),HERV-K109 (AF164615), HERV-K113 (AY037928.1), HERV-K115 (AY037929.1),and HML-2.HOM (AF074086.2).

Various procedures known in the art may be used for the production ofpolyclonal antibodies to polypeptides, derivatives, or analogs. For theproduction of antibody, various host animals, including but not limitedto rabbits, mice, rats, sheep, goats, etc, can be immunized by injectionwith the polypeptide or a derivative (e.g., fragment or fusion protein).The polypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum.

Monoclonal antibodies directed toward a HERV-K env polypeptide,fragment, analog, or derivative thereof, may be prepared by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture may be used. These include but are notlimited to the hybridoma technique originally developed by Kohler andMilstein Nature 256:495-497, 1975), as well as the trioma technique, thehuman B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72,1983; Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030, 1983),and the EBV-hybridoma technique to produce human monoclonal antibodies(Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96, 1985). Accordingly, the present disclosure is alsodirected to hybridoma cell lines that produce a monoclonal antibody thatspecifically binds to an antigen (e.g., HERV-K env protein) of a HERV-K⁺cancer.

Additionally, “Chimeric antibodies” may be produced (Morrison et al., J.Bacteriol. 159:870, 1984; Neuberger et al., Nature 312:604-608, 1984;Takeda et al., Nature 314:452-454, 1985) by splicing the genes from anon-human antibody molecule specific for a polypeptide together withgenes from a human antibody molecule of appropriate biological activity.For example, a chimeric antibody, wherein the antigen-binding site isjoined to human Fc region, e.g., IgG1, may be used to promoteantibody-dependent mediated cytotoxicity or complement-mediatedcytotoxicity. In addition, recombinant techniques known in the art canbe used to construct bispecific antibodies wherein one of the bindingspecificities is that of an antibody of the present disclosure (See,e.g., U.S. Pat. No. 4,474,893).

According to the present disclosure, techniques described for theproduction of single chain antibodies (U.S. Pat. Nos. 5,476,786;5,132,405; and 4,946,778) can be adapted to produce HERV-K env proteinantigen-specific single chain antibodies. An additional embodiment ofthe disclosure utilizes the techniques described for the construction ofFab expression libraries (Huse et al., Science, 246:1275-1281, 1989) toallow the rapid and easy identification of monoclonal Fab fragments withthe desired specificity, or fragment derivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan also be generated by known techniques. For example, such fragmentsinclude, but are not limited to, the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.Anti-idiotypic monoclonal antibodies to the antibodies of the presentdisclosure are also contemplated.

In the production and use of antibodies, screening for or testing withthe desired antibody can be accomplished by techniques known in the art,e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In a specific embodiment, antibodies of the present disclosure areconjugated to a secondary component, such as, for example, a smallmolecule, polypeptide, or polynucleotide. The conjugation may beproduced through a chemical modification of the antibody, whichconjugates the antibody to the secondary component. The conjugatedantibody may allow for targeting of the secondary component, such as,for example, a cytotoxic agent or an anti-tumor agent or an imagingagent, to the site of interest. The secondary component may be of anysize or length. Examples of secondary components include, but are notlimited to, chemotherapeutic agents, toxins, photo-activated toxins(e.g., dihydropyridine- and omega-conotoxin), radioactive isotopes,mitotic inhibitors, cell-cycle regulators, and anti-microtubuledisassembly compounds (e.g., taxol). For example, suitable cytotoxicagents include ricin A chain, abrin A chain, modeccin A chain, gelonin,melphalan, bleomycin, adriamycin, daunomycin, pokeweed antiviralproteins (PAP, PAPII, PAP-S), and granzyme B; and suitable anti-tumoragents include a lymphokine or oncostatin. In a specific embodiment, thesecondary component is the toxin Gelonin (rGel), which is a potentinhibitor of cellular protein synthesis. For example, rGel may be fusedto anti-HERV-K single-chain antibody (scFv) to produce a novel fusionprotein, namely HERV-K scFv/rGel.

Those skilled in the art will realize that there are numerousradioisotopes and chemocytotoxic agents that can be coupled to tumorspecific antibodies by well known techniques, and delivered tospecifically destroy tumor tissue. See, e.g., U.S. Pat. No. 4,542,225.Examples of imaging and cytotoxic reagents that can be used include¹²⁵I, ¹¹¹In, ¹²³I, ^(99m)Tc, ³²P, ³H, and ¹⁴C; fluorescent labels suchas fluorescein and rhodamine, and chemiluminescers such as luciferin.The antibody can be labeled with such reagents using techniques known inthe art, for example, as described in Wenzel and Meares,Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y. (1983) andColcer et al., Methods Enzymol., 121:802-16, 1986, and MonoclonalAntibodies for Cancer Detection and Therapy, Baldwin et al. (eds), pp.303-16 (Academic Press 1985).

Other covalent and non-covalent modifications of the antibodies orantibody fragments of the present disclosure are embraced herein,including agents which are co-administered or administered after theantibody or fragments, to induce growth inhibition or killing of thecells to which the antibody or fragment has previously bound.

In another embodiment of the present disclosure, compositions areprovided that comprise the monoclonal antibody, or antibody bindingfragment as described herein, bound to a solid support. A solid supportfor use in the present disclosure will be inert to the reactionconditions for binding. A solid phase support for use in the presentdisclosure must have reactive groups or activated groups in order toattach the monoclonal antibody or its binding partner thereto. Inanother embodiment, the solid phase support may be a usefulchromatographic support, such as the carbohydrate polymers SEPHAROSE®,SEPHADEX®, or agarose. As used herein, a solid phase support is notlimited to a specific type of support. Rather, a large number ofsupports are available and are known to one of ordinary skill in theart. Solid phase supports include, for example, silica gels, resins,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels, magnetic beads, membranes (including, but not limited to,nitrocellulose, cellulose, nylon, and glass wool), plastic and glassdishes or wells, and the like.

Antisense Constructs

The present disclosure provides antisense nucleic acids (includingribozymes and siRNAs), which may be used to inhibit expression of HERV-Kenv protein, particularly to suppress any effects on cell proliferation.An “antisense nucleic acid” is a single stranded nucleic acid moleculeor oligonucleotide which, on hybridizing under cytoplasmic conditionswith complementary bases in an RNA or DNA molecule, inhibits thelatter's role. If the RNA is a messenger RNA transcript, the antisensenucleic acid is a countertranscript or mRNA-interfering complementarynucleic acid. As presently used, “antisense” broadly includes RNA-RNAinteractions, RNA-DNA interactions, ribozymes, and RNase-H mediatedarrest. Antisense nucleic acid molecules can be encoded by a recombinantgene for expression in a cell (e.g., U.S. Pat. Nos. 5,814,500 and5,811,234), or alternatively they can be prepared synthetically (e.g.,U.S. Pat. No. 5,780,607). Also contemplated are vectors which includethese oligonucleotides or antisense constructs, for example,HERV-K1/1267 and K2/1267 lentiviral vectors.

Assay Systems

Any cell assay system that allows for assessing the presence of a HERV-Kenv protein is contemplated by the present disclosure. The assay may beused to screen for compounds that inhibit or prevent proliferation of aHERV-K⁺ cancer cell. For example, such assays may be used to identifycompounds that interact with a HERV-K env protein to regulatetranscription and translation, which can be evaluated by assessing theeffects of a test compound. In particular embodiments, changes inexpression of unique splice variants of HERV-K may be used to monitorthe effectiveness of test compounds that inhibit or prevent HERV-K⁺cancer cell proliferation.

Any convenient method permits detection of the expressed product. Forexample, the disclosure provides Northern blot analysis for detectingHERV-K env mRNA product. The methods comprise, for example, the steps offractionating total cellular RNA on an agarose gel, transferring RNA toa solid support membrane, and detecting a DNA-RNA complex with a labeledDNA probe, wherein the DNA probe is specific for a particular nucleicacid sequence of HERV-K env under conditions in which a stable complexcan form between the DNA probe and RNA components in the sample. Suchcomplexes may be detected by using any suitable means known in the art,wherein the detection of a complex indicates the presence of HERV-K envprotein in the sample.

Typically, immunoassays use either a labeled antibody or a labeledantigenic component (e.g., that competes with the antigen in the samplefor binding to the antibody). Suitable labels include without limitationenzyme-based, fluorescent, chemiluminescent, radioactive, or dyemolecules. Assays that amplify the signals from the probe are alsoknown, such as, for example, those that utilize biotin and avidin, andenzyme-labelled immunoassays, such as ELISA assays.

For in vitro assay systems, test compounds may be added to cell culturesof host cells, prepared by known methods in the art, and the level ofHERV-K env protein mRNA and/or protein are measured. Various in vitrosystems can be used to analyze the effects of a test compound on HERV-Kenv protein transcription and translation.

Nucleic Acid Assays

The DNA may be obtained from any cell source. DNA is extracted from thecell source or body fluid using any of the numerous methods that arestandard in the art. It will be understood that the particular methodused to extract DNA will depend on the nature of the source. Generally,the minimum amount of DNA to be extracted for use in the presentdisclosure is about 25 pg (corresponding to about 5 cell equivalents ofa genome size of 4×10⁹ base pairs). Sequencing methods are well known inthe art.

In another alternate embodiment, RNA is isolated from biopsy tissueusing standard methods well known to those of ordinary skill in the artsuch as guanidium thiocyanate-phenol-chloroform extraction (Chomocyznskiet al., Anal. Biochem., 162:156, 1987). The isolated RNA is thensubjected to coupled reverse transcription and amplification bypolymerase chain reaction (RT-PCR) or real time RT-PCR, using specificoligonucleotide primers that are specific for a selected site.Conditions for primer annealing are chosen to ensure specific reversetranscription and amplification; thus, the appearance of anamplification product is diagnostic of the presence of a particulargenetic variation. In another embodiment, RNA is reverse-transcribed andamplified, after which the amplified sequences are identified by, e.g.,direct sequencing. In still another embodiment, cDNA obtained from theRNA can be cloned and sequenced to identify a mutation.

In a specific embodiment, the presence or absence of a HERV-K⁺ cancer ina patient may be determined by evaluating the level of mRNA encoding aHERV-K env polypeptide within the biological sample (e.g., a biopsy,mastectomy and/or blood sample from a patient) relative to apredetermined cut-off value. Such an evaluation may be achieved usingany of a variety of methods known to those of ordinary skill in the artsuch as, for example, in situ hybridization and amplification bypolymerase chain reaction.

Protein Assays

In an alternate embodiment, biopsy tissue is obtained from a subject.Antibodies that are capable of specifically binding to HERV-K envprotein are then contacted with samples of the tissue to determine thepresence or absence of a HERV-K env polypeptide specified by theantibody. The antibodies may be polyclonal or monoclonal, preferablymonoclonal. Measurement of specific antibody binding to cells may beaccomplished by any known method, e.g., quantitative flow cytometry,enzyme-linked or fluorescence-linked immunoassay, Western analysis, andthe like.

Immunoassay technology, e.g., as described in U.S. Pat. Nos. 5,747,274and 5,744,358, and particularly solid phase “chromatographic” formatimmunoassays, are preferred for detecting proteins in blood or bloodfractions.

Diagnostic Tests

The antibodies of the present disclosure are also useful for diagnosticapplications, both in vitro and in vivo, for the detection HERV-K envprotein and HERV-K⁺ cancers, for example, breast cancer, ovarian cancer,and melanoma. Therefore, one embodiment of the present disclosure isdirected to the detection and/or measurement of HERV-K env protein in asample and the use of such detection or measurement in the diagnosis,staging, determination of severity, and prognosis in general of thedisorder.

In vitro diagnostic methods include immunohistological detection oftumor cells (e.g., on human tissue cells for excised tumor specimens),or serological detection of tumor-associated antigens (e.g., in bloodsamples or other biological fluids). Immunohistochemical techniquesinvolve staining a biological specimen such as tissue specimen with theantibody of the disclosure and then detecting the presence of antibodycomplexed to its antigen as an antigen-antibody complex. The formationof such antibody-antigen complexes with the specimen indicates thepresence of multiple ovarian, melanoma, or breast cancer cells in thetissue. Detection of the antibody on the specimen can be accomplishedusing techniques known in the art such as immunoenzymatic techniques,e.g., immunoperoxidase staining technique, or the avidin-biotintechnique, or immunofluorescence techniques.

Serologic diagnostic techniques involve the detection and quantificationof tumor-associated antigens that have been secreted or “shed” into theserum or other biological fluids of patients thought to be sufferingfrom multiple myeloma. Such antigens can be detected in the body fluidsusing techniques known in the art such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbant assays (ELISA) wherein antibody reactivewith the shed antigen is used to detect the presence of the antigen in afluid sample.

In a particular embodiment, the diagnostic techniques described can beused to follow the progress of therapy. In a subject undergoingtherapeutic treatment that results in an increase or a decrease in theamount of HERV-K env protein bearing cells, the amount of HERV-K envprotein bearing cells in a sample may serve as a useful measure for thesuccess or failure of the treatment. Thus, the present disclosureprovides a method for monitoring the effect of a therapeutic treatmentin a subject which comprises measuring at suitable time intervals theamount of HERV-K env protein expressed in a sample of tissue suspectedof containing HERV-K env protein expressing cells. The total amount ofHERV-K env protein is compared to a baseline or control value whichdepending on the disease, and the treatment, may be the amount of HERV-Kenv protein in a similar sample from a normal subject, from the patientprior to disease onset or during remission of disease, or from thepatient prior to the initiation of therapy. One of ordinary skill in theart will readily discern the appropriate baseline value to use in aparticular situation without undue experimentation.

Any procedure known in the art for the measurement of analytes can beused in the practice of the measurement of HERV-K env protein in asample using the compounds of the present disclosure. Such proceduresinclude but are not limited to competitive and non-competitive assaysystems using techniques such as radioimmunoassays, enzyme immunoassays(EIA), preferably the enzyme linked immunosorbent assay (ELISA),“sandwich” immunoassays, precipitin reactions, gel diffusion reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, immunoelectrophoresis assays, and the like.

For diagnostic and prognostic applications, a compound of the presentdisclosure, typically a hybrid molecule as described above will belabeled with a detectable moiety and used to detect HERV-K env proteinin a sample. Numerous labels are available which can be preferablygrouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The hybridmolecules can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, Volumes 1 and 2, Coligenet al., Ed., Wiley-Interscience, New York, N.Y., Pubs., (1991) forexample and radioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the hybrid molecules using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme preferablycatalyses a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Examples ofenzymatic labels include luciferases (e.g., firefly luciferase andbacterial luciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, Methods in Enzym. (ed J. Langone & H. Van Vunakis),Academic press, New York, 73: 147-166, 1981.

In the assays of the present disclosure also my use a solid phasesupport or carrier to which a hybrid molecule or an antigen is bound. By“solid phase support or carrier” is intended any support capable ofbinding an antigen or antibodies. Well-known supports, or carriers,include glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amyloses, natural and modified celluloses, polyacrylamides, agaroses,and magnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present disclosure. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, and the like.

Kits comprising one or more containers or vials containing componentsfor carrying out the assays of the present disclosure are also withinthe scope of the disclosure. For instance, such a kit can comprisereagents required for the immunohistochemical analysis of a sample suchas a tumor biopsy. Reagents may include one or more binding partners,e.g. a hybrid molecule or an antibody. For histological assays the kitcontains the chromogenic substrate as well as a reagent for stopping theenzymatic reaction when color development has occurred. The substrateincluded in the kit is one appropriate for the enzyme conjugated to oneof the hybrid molecules of the present disclosure. These are well-knownin the art. The kit can optionally also comprise a standard, e.g., aknown amount of purified HERV-K env protein.

Pharmaceutical Compositions

The present disclosure is also directed to pharmaceutical compositionscomprising a monoclonal antibody, or binding fragment thereof, whichspecifically binds to a HERV-K env protein, together with apharmaceutically-acceptable carrier, excipient, or diluent. Suchpharmaceutical compositions may be administered in any suitable manner,including parental, topical, oral, or local (such as aerosol ortransdermal) or any combination thereof. Suitable regimens also includean initial administration by intravenous bolus injection followed byrepeated doses at one or more intervals.

Pharmaceutical compositions of the compounds of the disclosure areprepared for storage by mixing a peptide ligand containing compoundhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 18th ed., 1990), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The compositions herein may also contain more than one active compoundsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Alternatively, or in addition, the composition may comprise acytotoxic agent, cytokine, growth inhibitory agent and/orcardioprotectant. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

Vaccines may comprise one or more such compounds in combination with animmunostimulant, such as an adjuvant or a liposome (into which thecompound is incorporated). An immunostimulant may be any substance thatenhances or potentiates an immune response (antibody and/orcell-mediated) to an exogenous antigen. Examples of immunostimulantsinclude adjuvants, biodegradable microspheres (e.g., polylacticgalactide) and liposomes (into which the compound is incorporated; seee.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccine preparation isgenerally described in, for example, M. F. Powell and M. J. Newman,eds., Vaccine Design (the subunit and adjuvant approach), Plenum Press(NY, 1995). Pharmaceutical compositions and vaccines within the scope ofthe present disclosure may also contain other compounds, which may bebiologically active or inactive. For example, one or more immunogenicportions of other tumor-associated antigens may be present, eitherincorporated into a fusion polypeptide or as a separate compound, withinthe composition or vaccine. Humoral or cellular immune responses againsttumor-associated antigen may provide a non-toxic modality to treatcancer. The presence of these antigens is also associated with bothspecific CD4⁺ and CD8⁺ T cell responses. The pharmaceutical compositionsand vaccines within the scope of the present disclosure may capitalizeon these responses to increase their clinical benefit.

Alternatively, a vaccine may contain DNA encoding one or more of thepolypeptides as described above, such that the polypeptide is generatedin situ. In such vaccines, the DNA may be present within any of avariety of delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacteria and viral expressionsystems. Appropriate nucleic acid expression systems contain thenecessary DNA sequences for expression in the patient (such as asuitable promoter and terminating signal). Bacterial delivery systemsinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface. In a preferred embodiment, the DNA maybe introduced using a viral expression system (e.g., vaccinia or otherpox virus, retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. The DNA may also be “naked,” as described,for example, in Ulmer et al., Science 259:1745-1749 (1993), and reviewedby Cohen, Science 259:1691-1692 (1993). The uptake of naked DNA may beincreased by coating the DNA onto biodegradable beads, which areefficiently transported into the cells.

Any of a variety of immunostimulants may be employed in the vaccines ofthis disclosure. For example, an adjuvant may be included. Mostadjuvants contain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Suitable adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFNγ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

Therapeutic Methods

The antibodies or binding fragments of the present disclosure may beused without modification, relying on the binding of the antibodies orfragments to the surface antigen(s) of HERV-K⁺ cancer cells in situ tostimulate an immune attack thereon. Alternatively, the aforementionedmethod may be carried out using the antibodies or binding fragments towhich a cytotoxic agent is bound. Binding of the cytotoxic antibodies,or antibody binding fragments, to the tumor cells inhibits the growth ofor kills the cells.

As mentioned above, HERV-K env protein may serve as a tumor-associatedantigen which can be used to elicit T cell and B cell responses. Intherapeutic applications, this may be used to reduce immune tolerancein, for example, a cancer patient. For example, HERV-K env protein isexpressed on both the cell surface and cytoplasm of breast cancer cells,therefore providing a target for both B cells and T cells, andpotentially greatly increasing the effectiveness of HERV-K as atumor-associated antigen.

Autologous DCs pulsed with HERV-K env protein enables autologousprofessional antigen presenting cells to process and present one or moreHERV-K epitopes in association with host human leukocyte antigen (HLA)molecules. Accordingly, in particular embodiments a therapeutic methodof the present invention comprises pulsing autologous DCs with HERV-Kenv protein to treat a HERV-K⁺ cancer. In general, DCs pulsed withHERV-K env protein induce T cell responses, enhance granzyme Bsecretion, induce CTL responses, and increase the secretion of several Thelper type 1 and 2 cytokines.

In particular embodiments, antibodies specific for HERV-K env proteinmay be used in conjunction with other expressed HERV antigens. This maybe particularly useful for immunotherapy and antibody treatments ofdiseases in which several different HERVs are expressed. For example,HERV-E in prostate, ERV3, HERV-E and HERV-K in ovarian cancer, and ERV3,HERV-H, and HERV-W in other cancers.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

EXAMPLES

To facilitate a better understanding of the present disclosure, thefollowing examples of specific embodiments are given, which are providedby way of exemplification and not by way of limitation.

Example 1 Expression of HERV-K Env Surface Proteins in Breast TumorEpithelial Cells

Since protein expression is a prerequisite for generating an immuneresponse, we first confirmed the presence and localization of HERV-K envprotein expression in breast cancer tissues by immunohistochemistryusing anti-HERV-K-specific antibodies. Expression of HERV-K surface envprotein was detected in breast tumor epithelial cells (more than 85% ofbreast tumor epithelial cells are HERV-K⁺), but not in normal oruninvolved breast epithelial cells (more than 90% of uninvolved breastepithelial cells are HERV-K⁻). Representative examples obtained from abreast biopsy are shown in FIG. 1A. FIG. 1A shows the detection ofHERV-K env protein expression in tumor epithelial cells obtained from apatient with infiltrating ductal carcinoma. Serial breast tissuesections obtained from a breast cancer patient were assessed byimmunohistochemistry using antibody specific against HERV-K env protein.The expression of HERV-K env protein was detected only in tumorepithelial cells, including ductal carcinoma in situ (DCIS) and invasiveductal carcinoma (IDC), but not in uninvolved normal epithelial cellsobtained from the same tumor tissue section (Normal epithelial cells)(C).

We also compared the expression of HERV-K env protein in multiple tissuemicroarray slides under identical staining conditions. The slides wereassigned a score of “0” to indicate no expression, “1” to indicate lowexpression, “2” to indicate intermediate expression, and “3” to indicatestrong expression of HERV-K env protein. Examples of the stainingpatterns observed in tissue-microarray slides are shown in FIG. 1B. InFIG. 1B, Case #1 was a normal mammary lobule from a 43-year-old female;case #4 was a normal mammary lobule from a 50-year-old female; case #16was a mammary gland tissue from a 61-year-old female; case #8 was a IDC(grade II) from a 45-year-old female; case #17 was a IDC (grade II; 49year-old female); Case #11 was a intraductal carcinoma (grade II) from a52-year-old female.

The three normal breast tissue samples shown in FIG. 1B (Case #1, #4,and #16) did not express HERV-K env protein; whereas, the three breastcancer tissues (Case #8, #11, and #17) had intermediate or strongexpression of HERV-K env protein. The expression profiles of HERV-K envprotein in the tissue microarray are summarized in FIG. 1C. The amountof HERV-K expression increased during the progression from normal tobenign to cancerous (Table 1). Fisher's exact test shows expressionlevels are associated with tumor tissue types (e.g., cancer, benign, andnormal) with P value <0.001. Specifically, all the cancer tissuesexpressed HERV-K protein with the majority (96%) displaying moderate orstrong expression. In contrast, 92% of normal and benign tissues had noexpression in HERV-K protein.

BC biopsies (case # 17 and 11) had intermediate or strong expression ofHERV-K env protein, whereas the normal breast tissue sample (case 4) didnot express HERV-K env protein. The expression profiles of HERV-K envprotein in the tissue microarray (N=182) are summarized in FIG. 1D. Theamount of HERV-K expression increased during progression from normal(N=56) to benign (ductal epithelial hyperplasia; N=7) to cancerous(N=119), and levels of expression were significantly associated withtumor tissue type (cancer, benign, or normal) (P<0.001; Chi-squaretest). Overall, 85% of cancer tissues expressed HERV-K protein, with 63%displaying moderate or strong expression, while 92% of normal and benigntissues lacked HERV-K protein expression.

TABLE 1 The frequency of expression of HERV-K env protein in126-breast-tissue microarrays Total Age 0¹ 1² 2³ 3⁴ Cancer⁵ 70 39.29  0⁶3 (4.29%) 16 (22.86%) 51 (72.86%) Normal⁷ 44 48.71 42 (95.45%) 2 (4.55%)Benign⁸ 7 54.8  5 (71.43%) 2 (28.57%) P value⁹ <0.001 ¹”0” no expression²”1” is low expression ³”2” is moderate expression ⁴”3” is strongexpression ⁵Cancer, infiltrating ductal carcinoma ⁶Values are numbers ofsamples in a given category, with percentage in parentheses ⁷Normal,normal breast tissues ⁸Benign, benign breast disease tissues ⁹P valuecalculated using Fisher exact test

TABLE 2 Patient characteristics Patient sample Lymph node Date of numberAge Diagnosis status Diagnosis C1 51 IDC¹ 12/1992  C2 59 IDC 4/2004 C340 DCIS + ILC — 6/2004 C4 54 DCIS + IDC 3/2003 C5 41 IDC stage 3 9/2004C6 41 IDC — 4/2004 C7 49 IDC — 1/2000 C8 55 DCIS — 6/2005 C9 32 IDC —10/2003  C10 55 IDC + Colon C positive 6/2005 C11 38 IDC positive 6/2005C12 49 DCIS — 6/2005 C13 42 DCIS — 11/2005  C14 79 IDC — 1994 ¹IDC,Infiltrating ductal carcinoma; DCIS, ductal carcinoma in situ; ILC,invasive lobular carcinoma; Colon C, colon cancer

In our study, the expression of both types of HERV-K, including envtranscripts (FIG. 1 and Table 5), spliced subgenomic env transcripts(FIG. 2, Table 1 and 2), and full-length HERV-K transcripts (FIG. 3 andTable 5), was detected in the majority of breast cancer tissues. We arethe first report that both types of HERV-K env transcripts were capableof being spliced into subgenomic env transcripts, and various splicedonor and acceptor sites (Table 5 and 6; FIG. 4) were detected in breastcancer tissues.

TABLE 5 cDNA sequence analysis of a breast cancer sample (invasiveductal carcinoma) Transcripts Primer used Clone No. Homology HERV-KAccession nt Env region K1-5′&K1-3′ 165K10C3 100% K102 AF164610.16491-7552 (type 1) K1-5′&K1-3′ 165AC4E 99% K102 AF164610.1 6491-7575K1-5′&K1-3′ 165KC2 99% K102 AF164610.1 6471-7575 Env region K2-5′&K1-3′165K22EC 98% K109 AF164615.1 6660-7501 (type 2) K2-5′&K1-3′ 165K22C1197% K (I) AB047209.1 7533-8680 K2-5′&K1-3′ 165K22C18 98% K (I)AB047209.1 7533-8680 K2-5′&K1-3′ 165K22C6 98% K109 AF164615.1 6659-7194Full-Length P1&P3 165P1C2 98% K102 AF164610.1 6072-7690 (type 1) P1&P3165P1C8 95% K (II) AB047240.1 7016-8632 Spliced U5 &Env A 165U2 99% cORFX82271.1  12-533 subgenomic U5 &Env A 165U3 99% K102 AF164610.1 833-878/5997-6658 type 1 & U5 &Env A 165U4 99% cORF X82271.1  12-533type 2 U5 &Env A 165U5 99% K102 AF164610.1  835-878/5997-6658 U5 &Env B165EBC22 99% K102 AF164610.1 835-1076/8117-8186 U5 &Env B 165EBC23 99%K102 AF164610.1 835-1076/8117-8186

TABLE 6 Analysis of splice donor (SD) and acceptor (SA) in breast cancercell lines and tissues Tissue 165U2¹ 165U3² 165U4¹ 165U5² 177U26¹177U29¹ 178U11² 178U15² 165UB22 165UB23 SD 1076  876 1076  876  961  961 927  927 1076 1076 SA 6433 5997 6433 5997 6948 6948 6399 6399 8117 8117ORF⁶ √ √ √ √ √ √ √ √ √ √ Cell lines⁹ T47DU3² T47DU4² MCF7c28³ 231U3³ZR75c33⁸ Tera2U2² Tera2U3² Tera1U13¹ Tera1U15¹ Tera1UB4¹ SD  883  8832076 2076 1076 1076 1076 1076 1035 1076 SA 6222 6222 7599 7599 8410 64336433 6433 6756 6433 2^(nd) SD⁴ 6492 6492 2^(nd) SA⁵ 6946 6794 ORF⁶ √ √N/A⁷ N/A N/A X X √ X √ ¹Type 2 HERV-K, nt numbered according toHML-2.HOM sequence (AF074086.2). ²Type 1 HERV-K, nt numbered accordingto HERV-K102 sequence (AF164610). ³Type 1 HERV-K, nt numbered accordingto HERV-K (II) sequence (AB047240). ⁴second splice donor. ⁵second spliceacceptor. ⁶ORF: Open reading frame. √ means ORF without stop codon, andX means ORF with one or more than one stop codon. ⁷N/A: could not bedetermined. ⁸Type 2 HERV-K, nt numbered according to HERV-K113 sequence(AY037928.1). ⁹231: MDA-MB-231; ZR75: ZR75-1.

Example 2 Detection of Anti-HERV Antibodies in Sera of Breast CancerPatients

We assayed for anti-HERV antibodies in sera from patients with variouscancers including breast cancer. ELISA analysis was used to determinethe binding affinity and specificity of antibodies in the sera obtainedfrom breast cancer patients (N=48) and normal female controls (N=50).The cutoff value was 0.5 for optical density at 405 nm. Approximately50% of the 48 breast cancer patient samples were positive for antibodiesagainst HERV-K env surface protein, 15% were positive for antibodiesagainst HERV-K gag protein, and 35% were positive for antibodies againsta splice variant of the env protein. In contrast, no anti-HERV-Kantibodies were detected in the control samples. ELISA data obtainedfrom 10 breast cancer and 5 control samples are shown in FIG. 2A. Thepresence of anti-HERV-K env protein antibodies, including anti-HERV-Ksurface env protein antibody (K-SU), anti-HERV-K gag protein (K-gag),and anti-HERV-K spliced env protein (Kspliced), provides indirectevidence of the presence of HERV-K env proteins in human breast cancer.We further tested for detection of potential serum IgG antibody againstHERV-K in cancer patients or control subjects by ELISA. The proportionof seropositive patients whose tumors were HERV-K positive was higherthan the proportion in control subjects without cancer (P=0.03; FIG.2B).

Example 3 Phenotyping of Immature and Mature Human DCs

DCs were generated from PBMCs cultured in medium containing thecytokines GM-CSF and IL-4. Immature DCs were exposed to TNF-α overnightfor maturation, with or without prior pulsing with HERV-K proteins.Characteristically, HERV-K-pulsed mature DCs showed enhanced CD83expression, relative to immature DCs and mature DCs treated with TNF-αonly. Expression of CD83+/CD209+ and CD83+/CD86+ was also higher inHERV-K pulsed mature DCs than in immature DCs and mature DCs (FIG. 3A).

We determined that HERV-K env protein is expressed in HERV-K pulsed DCs,by flow cytometry using anti-HERV-K antibody or anti-RGS mAb. More than50% or 70% of DCs pulsed with HERV-K env surface protein became HERV-Kantigen positive cells as assessed using anti-HERV-K or anti-RGS mAb,respectively (FIG. 3B).

Example 4 Surface Expression of HERV-K Env Protein on Breast CancerCells Lines

No report to date has provided direct evidence of the surface expressionof HERV-K env protein in any non-transfected cell line. We thereforeexamined the expression of this protein on the surface and in thecytoplasm of breast cancer cells by flow cytometric analysis andfluorescence microscopy of cells stained with antibody against HERV-Kenv protein. Surface expression of HERV-K env protein was observed onnonpermeabilized malignant MCF-7 (50% positive surface expression) andT47D (25%) breast cancer cell lines, and cytoplasmic expression (95% ofMCF-7 and 95% of T47D cells) was observed in cells permeabilized byTriton X-100 flow cytomety. In contrast, no surface or cytoplasmicexpression of HERV-K was observed in benign MCF10A or premalignantMCF10AT breast epithelial cells (1% to 3% surface expression and 3% to5% cytoplasmic expression, respectively). The observation was furtherconfirmed by fluorescence microscopy (FIG. 3C).

Using immunofluorescence microscopy, we found HERV-K env proteinexpression on the surface of non-permeabilized BC cell lines such asMCF-7, T47D, MDA-MB-231 BC cells as well as in the cytoplasm followingpermeabilization with Triton X-100 (FIG. 16A; MDA-MB-231 cells).However, HERV-K env protein expression was not observed in benign MCF10Aor premalignant MCF10AT breast epithelial cells under these conditions(data not shown). Surface expression of HERV-K env protein on MCF-7(FIG. 16B), but not on MCF-10A cells (FIG. 16C), was detected usingconfocal microscopy and the percentage of surface expression in thecells was determined by flow cytometric analysis (FIG. 16D). HERV-K envprotein was detected in several BC cell lines, but not in MCF-10A andMCF-10AT non-cancer breast cells (FIG. 16E) by Western blot usinganti-K-SU antibody.

Example 5 T-Cell Proliferation Assay

Non-IVS PBMCs or CD3+ cells obtained from normal donors (N=7) werestimulated with HERV-K or HERV-E env surface protein and tested foractivation of human T cell proliferation. We found that HERV-K or HERV-Eenv surface protein does not stimulate human T cell proliferation in amanner similar to the bone fide superantigen Staphylococcal enterotoxinA (SEA; FIG. 4A).

We stimulated PBMCs with autologous DCs pulsed with HERV-K env antigen,matured the PBMCs to produce antigen specific 1-week or 3-week IVS andassessed the ability of the PBMCs to stimulate T-cell proliferation. Thefold-increase in proliferation of HERV-K specific T cells wassignificantly greater in IVS cells from cancer patients than in thosefrom normal female control subjects, compared with autologous PBMCs(FIG. 4B).

Example 6 Granzyme B ELISPOT Assays with IVS or PBMCs from CancerPatients

PBMCs (stimulated with unpulsed-DCs), 1-week IVS cells, or 3-week IVScells obtained from cancer patients and normal donors were used inELISPOT assays to quantitate cells producing granzyme B (GrB), animportant effector molecule of CTL and natural killer cells. GrB spotsdetected in 1-week or 3-week IVS were higher in cancer patients than innormal controls (FIG. 4C) (1-week IVS P=0.004; 3-week IVS P=0.003). Veryfew or no GrB spots were detected from PBMCs stimulated with unpulsedDCs obtained from normal donors or cancer patients.

Example 7 HERV-K Specific CTL Cytotoxic Responses

51Cr release cytotoxicity assay was used to assess the immunogenicity ofHERV-K from IVS cells obtained from control and cancer samples. HERV-Kspecific cytotoxicity resulted in 20% to 60% lysis of HERV-K expressingcells in the cancer patients only, with nonspecific cytotoxic activitybelow 15% (FIG. 4D). Natural killer (NK) cell activity was assessed bycytotoxicity against K562 cells, which are susceptible to NK cells. IVScells stimulated with HERV-K-pulsed DCs were capable of killingHERV-K-loaded autologous DCs or B-LCL cells, but not DCs or B-LCL cellsloaded with an irrelevant mock protein (human LMP2A, purified from thesame expression vector). HERV-K-specific IVS cells from cancer patientsthat were stimulated with HERV-K-pulsed DCs did not increase lysis ofK562 cells; whereas HERV-K-specific IVS cells obtained from normaldonors did increase cytoxicity toward K562 cells (data not shown). CTLsobtained from two breast cancer patients showed strong lytic activityagainst the HERV-K positive breast cancer cell line MCF-7. The highercytotoxicity against MCF-7, relative to HERV-K-loaded DCs or B-LCLcells, is probably due to the higher expression of the naturallyprocessed HERV-K proteins at the surface of MCF-7 cells relative to DCsor B-LCL cells (lower transfection efficiency), as assessed by flowcytometry with anti-HERV-K antibody (data not shown). Thus, HERV-K envprotein does not suppress NK cell responses, and the lack of suppressionwould provide a potential mechanism for breaking tolerance by the hostimmune system.

Example 8 Detection of Cytokines in HERV-K Specific IVS Cells

Cytokine bead array assays for human cytokines were used to detect thesecretion of cytokines by PBMCs treated with unpulsed DCs or IVS cells(PBMCs stimulated with DCs pulsed with HERV-K env surface proteins).IL-2 secretion (P=0.001) and IFN-γ secretion (P<0.001) were markedlyincreased in IVS obtained from cancer patients, when compared to thepatients' PBMCs (N=12). IL-2 secretion (P=0.021) and IFN-γ secretion(P=0.061) were greater in IVS than in PBMCs obtained from normal controlsubjects (N=11), but the increase was not as great as in cancerpatients. IL-2 (P=0.029; FIG. 5A), IFN-γ (p=0.028; FIG. 5B), IL-6(P=0.038; FIG. 5C), and IL-8 (P=0.016; FIG. 5D) secretion were higher inHERV-K-specific IVS cells obtained from cancer patients, than in IVScells from control subjects. TNF-α secretion was non-significantlyincreased (P=0.063) in HERV-K-specific IVS cells obtained from cancerpatients, relative to IVS cells from control subjects. IL-1β secretionor IL-4 secretion was unchanged in HERV-K-specific IVS cells obtainedfrom cancer patients, in comparison to IVS cells from control subjects.

Example 9 Intracellular Cytokine Expression by T Cells Specific forHERV-K Human Isotype Control

To evaluate whether TNF-α, IL-2, or IFN-γ expression are increased inHERV-K-stimulated IVS cells, intracellular cytokine expression wasassessed at the single-cell level. PBMCs treated with unpulsed DCs, orIVS cells obtained from the same PBMCs stimulated with HERV-K pulsedDCs, were left unactivated as negative controls, were nonspecificallyactivated with a leukocyte activation cocktail which included PMA,ionomycin and brefeldin A (positive controls; non-specific), or wereactivated with HERV-K plus brefeldin A (HERV-K-stimulated).Intracellular expression of TNF-α, IL-2, and IFN-γ by CD3+ T cells wasanalyzed by four-color flow cytometry. As shown in FIG. 6, activated IVScells had higher intracellular TNF-α and IFN-γ, but not IL-2, than theirautologous unactivated PBMCs. Similar results were consistently obtainedin several similar experiments. APC-labeled anti-IgG2a or anti-IgG1antibodies, and a PE-labeled isotype control cocktail did not staineither the activated or unactivated cells (data not shown).

Cytokine production after HERV-K antigen stimulation. Human cytokinebead array assays were used to detect the secretion of T-helper 1 (Th1)and Th2 in PBMC or IVS obtained from PBMC stimulated with DC pulsed withK-SU protein. The secretion of each cytokine was induced significantlyby antigen-pulsed DC over secretion levels of unpulsed control DC (datanot shown). We compared cytokine secretion in PBMC and IVS, in both BCpatients and normal donors. Secretion of a number of cytokines wasaltered after HERV-K stimulation. Secretion of the Th1 cytokines IL-2and IFN-γ was significantly enhanced in BC patient K-SU-stimulated IVScells, in comparison to K-SU-stimulated PBMC. IFN-γ secretion wassignificantly lower in BC patients than in normal donors before HERV-Kenv stimulation. A summary of IL-2 and IFN-γ secretion is presented inFIGS. 6C and 6D. Interestingly, elevated IP10 secretion was alsoobserved in cancer patients (data not shown). Other cytokines includingIL-6 and Il-8 were increased in BC patients (data not shown). We alsoanalyzed cytokine production ex vivo from freshly-isolated PBMC from BCpatients and normal healthy donors by intracellular cytokine staining(ICS). PBMC were activated with HERV-K-pulsed or unpulsed DC for 6 h andfixed and subjected to ICS. As shown in FIGS. 6A and 6B, a markedlyincreased frequency of TNF-α, IL-2 and IFN-γ-secreting CD8⁺ T cells weredetected in the HERV-K-stimulated cultures.

Thus, T cells isolated from BC patients exhibit HERV-K-specificproliferation, pro-inflammatory cytokine secretion, and cytotoxicactivity against HERV-K target cells not found in normal healthycontrols.

Materials and Methods for Examples 1-9

Synthesis of HERV-K env Fusion Proteins and Antibodies. HERV-K envsurface cDNAs obtained from breast cancer tissue, as described inWang-Johanning F, et al. Clin Cancer Res 7(6):1553-60 (2001), werecloned into the corresponding enzyme-digested QIA expression vector(pQE30; Qiagen; Valencia, Calif.), which contains a 6-His tag at theN-terminus. Colonies positive for HERV-K env expression were furtheridentified by restriction enzyme analysis and characterized bysequencing with vector-specific primers to confirm that the clonesproduced the desired HERV-K env proteins. The colonies confirmed bysequencing were induced with isopropyl-B-D-thiogalactopyranoside and theHERV-K env fusion proteins were purified by affinity chromatographyusing Ni-nitrilotriacetic acid (Ni-NTA; Qiagen) agarose for the pQEvector. HERV-E env surface cDNAs obtained from prostate cancer tissue,as described in Wang-Johanning F, et al. Cancer 98(1):187-97 (2003),were cloned into pQE30 to produce HERV-E env surface protein. Thepurified HERV env fusion proteins were used to immunize rabbits or micefor the production of polyclonal or monoclonal anti-HERV-K envantibodies, respectively, using standard techniques. The antibodies werefurther purified using Protein G Sepharose 4 Fast Flow (AmershamPharmacia Biosciences; Piscataway, N.J.) and tested for specificity andsensitivity against various HERV env proteins by enzyme-linkedimmunosorbent assay (ELISA) and/or immunoblot analysis.

HERV-K Expression in Breast Cancer Cell Lines, Breast Cancer PatientTissues, and Breast Tissue Microarrays. The human breast cell linesMCF-7, T47D, MCF-10A and MCF-10AT, as well as the human chronicmyelogenous leukemia cell line K562 and Epstein-Barr virus(EBV)-transformed tamarin cells B95-8, were obtained from the AmericanType Culture Collection (Rockville, Md.) and were cultured in the mediarecommended by the manufacturer. MCF10A cells were gifts obtained fromDr. Robert Pauley, and were cultured in his recommended media. Forimmunohistochemistry, formalin-fixed, paraffin-embedded tissues wereused. Two tissue microarrays, each with 63 breast tissue samples fromnormal women and women with benign, malignant, or other breast diseases,were obtained from US Biomax, Inc (Rockville, Md.). Human breast tissuesand peripheral blood mononuclear cells from breast cancer patients ornormal female controls (Table 7) were obtained from The University ofAlabama at Birmingham and The MD Anderson Cancer Center with approvalfrom both of the Institutional Review Boards.

Immunohistochemical Analysis of Multiple Tissue Microarray Slides.Immunohistochemistry was performed on tissue microarray slides using anLV-1 Autostainer universal staining system (DAKO; Carpinteria, Calif.)compatible with currently available reagents for the staining ofparaffin-embedded and frozen tissue sections. These multiple tissuemicroarray slides allowed us to compare the expression of HERV-K envsurface (SU) protein in multiple tissues under identical stainingconditions. Slides were incubated for 5 minutes with 3% H2O2, for 10minutes with horse serum, and for 30 minutes with anti-HERV-K envantibodies (1:750 dilution). This was followed by a 15-minute incubationwith horseradish peroxide (HRP)-conjugated anti-rabbit or anti-mouseimmunoglobulin G (IgG) secondary antibody (DAKO), a 5-minute incubationwith diaminobenzidine for color development, and a 5-minute incubationwith the counterstain hematoxylin.

ELISA. ELISA assays were used to detect various anti-HERV antibodies inhuman sera, as described previously in Wang-Johanning F, et al. CancerRes 58(9):1893-900 (1998). Briefly, a 96-well ELISA plate was coatedwith various HERV fusion proteins (10 μg per ml, 100 μl per well) inphosphate-buffered saline (PBS) and incubated overnight at 4° C. Theplate was then blocked for 1 hour with 5% nonfat dry milk (Sigma; St.Louis, Mo.) and 3% bovine serum albumin at room temperature. Human sera(diluted 1:200 with PBS) were added to the coated wells, and the platewas incubated overnight at 4° C. After washing six times with PBS-T (0.5ml of Tween 20 in 1000 ml of PBS), 100 μl of HRP-conjugated anti-humanIgG antibody (1:2000 dilution, Sigma) was added to each well of theplate to detect the serum antibody, followed by incubation for 1 hour atroom temperature. The plate was washed again with PBS-T, and color wasdeveloped using 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid(ABTS; Sigma). After 10 to 30 minutes, the absorbance of the plate wellswas measured on a microplate reader at 405 nm. Anti-human IgG antibodywas used for negative controls and anti-RGS monoclonal antibody (mAb)was used for positive controls (Qiagen Inc.; used to detect 6-Hisprotein produced from pQE30 vector). The cutoff value for a negativereaction is 0.5 OD at 405 nm. For determining anti-HERV-K antibodyisotope, anti-human IgG antibody (5 μg per ml) was coated on the plate,human plasma samples (1:100 dilution; 100 μl per well) were added,followed by incubation for 1 hour at room temperature. HERV-K surfaceenv fusion protein (10 μg per ml) was then added, followed by anti-RGSmAb (1:1,000 dilution), and then HRP conjugated-anti-mouse IgG (1:2,000dilution). Additionally, all samples were tested on two wells not coatedwith HERV-K fusion protein or anti-IgG mAb to define non-specificreactivity. The final ELISA value was calculated by subtracting thenon-specific reactivity mean absorbance from the sample triplicate meanabsorbance. To control for inter-assay variation, positive IgG controlsselected from a previous study were included in each plate and tested asdescribed. All ELISA analyses were performed at least three times foreach serum sample. The means of the threshold values were used for thefinal analysis, as described in Wang-Johanning F, et al. Cancer Res58(9):1893-900 (1998).

Preparation of DCs from Human PBMC. DCs were Generated from Adherent orCD14-positive PBMCs isolated by magnetic cell sorting with CD14MicroBeads (Miltenyi Biotec; Auburn, Calif.). The isolated cells wereincubated in AIM-V medium (Gibco Life Technologies; Gaithersburg, Md.)with 10% human AB serum (Gemini Bioproducts; Woodland, Calif.) in thepresence of interleukin (IL)-4 (1000 IU/ml; R&D Systems; Minneapolis,Minn.) and granulocyte macrophage colony-stimulating factor (GM-CSF;1000 IU/ml; R&D Systems; Minneapolis, Minn.) for 6 days. Culture mediawere changed after 3 days. Following the 6-day incubation period, theimmature DCs were harvested and transfected with HERV-K env surfaceprotein or control proteins by lipofection withN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate(DOTAP). After a four-hour incubation, cells were replenished with newmedia containing the pro-inflammatory cytokine tumor necrosis factor(TNF)-α (R&D Systems), and incubated for 18 additional hours to obtainmature DCs. The mature DCs were harvested, washed once and mixed withautologous PBMCs for IVS or for assays. CD3+ cells were separated fromPBMCs by magnetic cell sorting with an autoMACS separator (MiltenyiBiotech) using human CD3 beads according to the manufacturer'sinstructions. B95-8 culture supernatants containing the transformingstrain of Epstein-Barr virus EBV were used to establish the Blymphoblastoid cell lines (B-LCL). B-LCL express type 3 latency genes(EBNA-2 and LMP-1) and were maintained in RPMI medium 1640 with 10% FCS.

Phenotyping of Immature and Mature DCs and Determination of SurfaceExpression of HER V-K Surface Protein. Immature or mature DCs with orwithout prior pulsing with HERV-K env protein were phenotyped on day 7using a multicolor kit with CD86PE/CD209 PerCP-Cy5.5/CD83 APC kit (BDBiosciences; San Jose, Calif.). The DCs were stained with monoclonalantibodies for 30 minutes at room temperature and analyzed by flowcytometry. To confirm that the env-pulsed DCs had become HERV-Kspecialized antigen-presenting cells, expression of HERV-K env surfaceprotein in pulsed DCs was determined by flow cytometry using anti-HERV-Kenv antibody (1:200 dilution). As a positive control, anti-RGS mAb(1:200; Qiagen) was used to detect His-target protein produced from thepQE30 vector. The DCs were incubated with the primary antibodies at 4°C. for 30 minutes and then with anti-IgG-FITC secondary mAb (1:1000dilution) at 4° C. for 15 minutes. DCs stained with only secondaryantibody served as negative controls. After being washed with PBS, thecells were fixed with 3% paraformaldehyde in PBS. Samples were analyzedon a BD FACSCalibur™ system (BD Biosciences). After confirming thespecificity of anti-HERV-K antibody to HERV-K env protein, culturedbreast cells were incubated with the antibody as described above. Forstaining of permeabilized cells, the cells were treated with 0.1% TritonX-100 in PBS, and then incubated with primary and secondary antibodies.Controls samples were incubated with anti-IgG-FITC secondary antibody.

In Vitro Sensitization. DCs were pulsed with antigen and matured asdescribed above. Autologous PBMCs (1×10⁶ cells/ml) were added to theloaded DCs at a DC:PBMC ratio of 1:20. Cells were incubated for 6-7 daysin AIM-V medium (Gibco) containing 10% human AB serum (GeminiBioproducts), 1000 U/ml penicillin (Gibco), 1000 μg/ml streptomycin(Gibco), and 10 IU/ml IL-2 (eBioscience; San Diego, Calif.) to produce1-week IVS cells. The 1-week IVS cells were restimulated on day 14 withautologous DCs previously pulsed with antigen to produce 3-week IVScells. The enriched IVS cells were then assessed with proliferation orEnzyme-Linked ImmunoSPOT (ELISPOT) assays. As unstimulated controls forIVS, PBMCs were cultured under the same conditions but with nonpulsedDCs.

T-Cell Proliferation Assay. T cell proliferation was evaluated for PBMCand IVS cells. Autologous monocyte-derived DCs were loaded, matured, andadded to PBMCs at a DC:PBMC ratio of 1:20. These cultures were set up intriplicate wells of 96-well plates at 100,000 cells/well in RPMI mediumcontaining 10% (v/v) human AB serum. A new set of DCs was pulsed on day6 with experimental or control antigens, matured and added either to theIVS cultures (T cell proliferation assay with IVS) or to fresh PBMCs(assay without IVS). The cultures were incubated for 5 days at 37° C.Methyl-[3H]thymidine (ICN Biomedicals; Costa Mesa, Calif.) was added andthe cultures were incubated for another 18 hours. The cells were thencollected and the incorporation of [³H]thymidine into cells was measuredas an indicator of cell proliferation using a liquid scintillation βcounter. Results were expressed as counts per minute (CPM) per 1×10⁵splenocytes.

ELISPOT Assays. A granzyme B ELISPOT assay to detect and quantitativecytokine-secreting cells in response to antigen was performed usingcommercial kits (Biosource International; Camarillo Calif.), followingthe manufacturer's recommendations. The spots were evaluated using anautomated ELISPOT reader system (Carl Zeiss; Thornwood, N.Y.) with KSELISPOT Software 4.5+ (ZellNet Consulting). Only spots with fuzzy ordiffuse borders were scored as positive. Net frequencies of spot-formingcells were calculated.

Cytotoxic T-Lymphocyte Release Assay. To determine whether HERV-K envsurface proteins are potential targets for a breast tumor vaccine, CTLassays were performed in round-bottomed 96-well plates using a standard4-hour 51Cr-release assay, as described in Dolbier C L, et al. J BehavMed 24(3):219-29 (2001). Five thousand 51Cr-labeled target cells wereadded to serial dilutions of effector cells in effector to target cellratios (E/T) up to 20:1. K562 cells were used as target cells in thesame experiment to detect natural killer cell activity. After a 4-hourincubation at 37° C., 25 μl of the supernatants were collected andradioactivity was quantitated using a gamma counter. To blockcytotoxicity, effector cells were pre-incubated for 30 minutes at roomtemperature with an anti-human CD3 mAb (10 μg/ml; Ortho PharmaceuticalCorp; Raritan, N.J.).

Cytokine Bead Array Analysis. PBMCs or IVS cells from cancer patients ornormal control subjects were cultured with HERV-K env protein (10μg/ml), human papillomavirus 16 E6 protein (10 μg/ml; purified from thesame expression vector as control protein), concanavalin A (10 μg/ml;used as control protein), or no protein for 48 hours at 37° C. in a 5%CO₂ atmosphere, under the previously described conditions for evaluatingT cell proliferation. After incubation, the supernatants were collectedand stored at −20° C. for cytokine bead array analysis using a LINCOplexmultiplex immunoassay-based protein array system (LINCO Research; St.Charles, Mo.), which contains microspheres conjugated with mAbs specificfor target proteins. Triplicate samples of cell culture supernatants (25μl) were assayed for the human cytokines IL-1β, IL-2, IL-4, IL-6, IL-8,TNF-α, and interferon (IFN)-γ. Antibody-coupled beads were incubatedwith the supernatants (antigen) overnight at 4° C., and weresubsequently incubated with a biotinylated detection antibody at roomtemperature for 1 hour. Incubation with streptavidin-phycoerythrin wasperformed at room temperature for 30 minutes. Data were reported as themean±standard deviation (SD) of triplicate wells. A standard curveranging from 0 to 10,000 pg/ml, in which mean fluorescence intensity wasplotted against cytokine concentration, was generated from knownconcentrations of various cytokines using a Luminex 100 instrument,which employs fluorescent bead-based technology (Luminex Corporation;Austin, Tex.).

Intracellular Cytokine Staining. Cytokines produced by CD4+ and CD8+ Tcells, including TNF-α, IL-2, and IFN-γ, were assayed by cytokine flowcytometry as previously described in Martins S L, et al. Blood104(12):3429-36 (2004) and Komanduri K V, et al. Nat Med 4(8):953-6(1998). PBMCs and IVS cells were activated by incubation with HERV-K envprotein and GolgiPlus (BD Biosciences) at 37° C. in a humidified 5% CO₂atmosphere for 4 hours. Cells activated with a Leukocyte ActivationCocktail (BD Biosciences) served as positive controls; whereasunstimulated cells served as negative controls. After the 4-hourincubation, the cells were blocked for 15 minutes and stained withsurface antibodies against CD4, CD8, and CD3 (BD PharMingen; San Diego,Calif.) for 20 minutes. The activated cells were permeabilized with BDCytofix/Cytoperm buffer for subsequent intracellular staining withPE-conjugated TNF-α, IL-2, and IFN-γ (BD PharMingen). APC-labeledanti-IgG2a or IgG1, and a PE-conjugated isotype control cocktail wereused as single color controls. The samples were acquired and analyzed ona FACSCalibur system (BD Biosciences).

Statistical Analysis. Each assay was performed in triplicate andanalyzed using SigmaStat 3.0 software. To evaluate the differencesbetween the proportions of positive samples in the different groups,data were arranged in the form of two-by-two contingency tables andanalyzed by Fisher's exact test to calculate the odds ratios, 95%confidence intervals and P values. Differences among groups wereanalyzed by the Kruskal-Wallis One Way Analysis of Variance on Ranks.The mean, standard deviation (SD) and coefficient of variation werecalculated for sample triplicates. A P value <0.05 indicated asignificant difference among treatments.

Example 10 Expression of Multiple HERV Env Transcripts in Human OvarianCancer Cells and Tissues

The expression of env transcripts of type 1 (1,104 bp) and type 2 (1,194bp) HERV-K surface domains was detected in ovarian cancer cell lines(PA1, SKOV3, OVCA 429, OVCA 433, OVCAR3, DOV 13, and OVCA 420), but notin normal ovarian epithelial cells (NOE 113, 114, 116, and 119). Anexample of the RT-PCR results is depicted in FIG. 7A. HERV-K expressionwas not detected by RT-PCR in normal and uninvolved ovarian tissues.Some ovarian cancer tissues expressed only type 1 HERV-K env regiontranscripts, some expressed only type 2 and some expressed both types ofHERV-K env transcripts with the same or varying intensities (data notshown). The expression of multiple HERV families, which included ERV3(1,744 bp), HERV-E (1,348 bp), and HERV-K types 1 and 2, was detected inovarian cancer tissues more than in matched uninvolved ovarian tissues(FIG. 7B). Expression of both of cORF (437 bp) and np9 (256 bp) RNA, aswell as full-length HERV-K env reading-frame transcripts, was detectedin ovarian cancer cells or some specimens (FIG. 7C).

Spliced np9 and/or cORF mRNA was expressed in ovarian cancer tissues andthe ovarian cancer cell lines DOV13, OVCAR 3, OVCA 429, and OVCA 433.Proteins cORF (type 2) located in the nucleus and with functions similarto the Rev protein of HIV (Boese A, et al. FEBS Lett 468:65-67, 2000;Tonjes R R, et al. J Acquir Immune Defic Syndr Hum Retrovirol13:S261-267, 1996), were shown in earlier studies to support celltransformation and to induce tumor formation in nude mice (Boese A, etal. Oncogene 19:4328-4336, 2000). Not wishing to be limited by theory ormechanism, splice variants may promote cell transformation, as wasobserved for cORF and np9; or the variants may inhibit the host immuneresponse, as was found when expression of a retroviral envelope proteinor transmembrane subunit led to tumor growth in vivo (Blaise S, et al.,2001). These results provide evidence that both types of HERV-K mRNA, aswell as multiple HERV family mRNAs, are transcribed in ovarian cancercell lines and tissues.

In order to quantitate the expression of HERV-K in human ovarianbiopsies, 254 ovarian tissue RNAs isolated from various ovarianspecimens were quantified for the expression of HERV-K env transcriptsby real time RT-PCR. The results of real-time RT-PCR analyses of thesesamples are presented in FIG. 7D. Lower CT values (HERV-K/S9 ratios)represent higher expression of HERV-K env transcripts. HERV-K envexpression was significantly greater in tissues from epithelial tumorwithout metastasis (p=0.012; N=121) and greater but not statisticallysignificant in tissues from epithelial tumor with metastasis (p=0.058;N=46), relative to expression in normal and benign ovarian tissues(N=19), using a two-sample t-test.

Example 11 Characterization of HERV-K Surface Env Fusion Protein andAnti HERV-K Antibody

Various HERV env cDNAs derived from cancer tissues were cloned in thebacterial expression vector systems pQE (with 6-His; Qiagen Inc.) andGST (Pharmacia). Some clones produced recombinant fusion proteins, suchas HERV-K-His fusion surface protein (40,000 daltons), HERV-K gag fusionprotein (84,460 daltons), and HERV-K env splice product protein(cORF-His; 14,000 daltons). Several other HERV env proteins have beenalso produced in our laboratory including ERV3 and HERV-E env proteins(data not shown). The authenticity of HERV env fusion proteins wasfurther confirmed by sequence analysis using vector specific primers.These purified HERV fusion proteins were used to detect the variousanti-HERV antibodies in human sera. These HERV fusion proteins were alsoused to produce antibodies. These positive antibodies were used to testfor their specificity or sensitivity against HERV env surface proteinsby ELISA analysis or Western blot analysis.

Example 12 Surface Expression of HERV-K Env Protein on Ovarian CancerCells Lines

Our data suggest that HERV-K env is expressed in ovarian cancer cellsand tissues at the transcriptional level. To evaluate the significanceof HERV-K env protein in ovarian cancer, we examined expression of thisprotein in cell surface and cytoplasmic compartments of ovarian cancercells by flow cytometric analysis of cells stained with anti-HERV-K envsurface protein specific antibody. Both cell surface and cytoplasmicexpression of HERV-K env protein was detected in ovarian epithelialcarcinoma cells including DOV13 (53% surface expression of HERV-K; FIG.8A; No-perm), SKOV3 (22%), OVCAR3 (25%), OVCA429 (16%), OVCA433 (15%),OVCA430 (16%), and OVCA420 cells (20%), but not in three normal ovariansurface epithelial cell lines. The observation was further confirmed byfluorescence microscopy (FIG. 8A). Surface expression of HERV-K envprotein was observed on nonpermeabilized malignant DOV13 and OVCA420ovarian cancer cell lines, and cytoplasmic expression was observed inDOV13 (FIG. 8A; Penn; 86%) and OVCA420 cells permeabilized by TritonX-100 by flow cytomety. In contrast, no surface or cytoplasmicexpression of HERV-K was observed in T29 or T80 epithelial cells,respectively.

Example 13 Expression of HERV-K Env Proteins in Ovarian Tumor EpithelialCells

Our data demonstrate the expression of HERV env transcripts in humanovarian cancer tissues. It is essential to test for HERV-K proteinexpression in these cancer tissues, because protein expression is aprerequisite for generating an immune response.

Multiple tissue microarrays TMA1, TMA2, and TMA3 contained 72, 85, and484 multiple ovarian tissues, respectively, and were stained with a DAKOautostainer universal staining system using anti-HERV-K env proteinantibody. A score of “0” indicates no expression, “1” indicates lowexpression and “2+3” indicates intermediate and strong expression ofHERV-K env protein, respectively. Examples of samples in TMA1 with 0, 1,2 and 3 scores after staining for HERV-K are shown in FIG. 8B. Normalovarian tissues had a “0” score, clear cell carcinoma had a score of“1”, serous papillary cystadenocarcinoma had a score of “2”, and serouspapillary adenocarcinoma had a score of “3”. The expression profiles ofHERV-K env protein detected from TMA1 are summarized in Table 3.Positive staining samples from TMA2 were mucinous cyst (FIG. 8C), lowmalignant potential, low-grade, high-grade (HG) endometrioid, serousLMP, LG serous, HG serous, and clear cell carcinoma.

TABLE 3 The expression profile of HERV-K env SU protein in ovariantissue microarray slide TMA1 containing 72 tissues. Histotype¹ ²Age(range) N 0 1 2 + 3 ³NL 45 (43-47) 3 3 (100%)⁴ ⁵GCT 35.2 (12-49) 5 3(60%) 2 (40%) ⁶Stroma T 49.5 (40-48) 4 4 (100%) ⁷Krukenberg T 46.6(24-66) 5 3 (60%) 1 (20%) 1 (20%) Brenner T 57 1 1 (100%) Borderline T75 1 1 (100%) ⁸Mets. AdCa 44.6 (30-70) 5 1 (20%) 4 (80%) ⁹Mucous P AdCa55.5 (42-69) 2 1 (50%) 1 (50%) Serous P AdCa 48.3 (22-66) 40 13 (33%) 10(25%) 17 (43%) Other carcinoma 47.7 (35-62) 3 2 (66.7%) 1 (33.3%) ¹⁰CCC43.6 (40-48) 2 2 (100%) Endometrioid C 40 1 1 (100%) Total 72 31 (43%)16 (19%) 28 (38%) ¹A score of 0 indicates no expression, 1 indicates lowexpression and 2 + 3 indicates intermediate and strong expression ²Age:Average age and range of ages of patients ³NL: Normal ovarian tissues⁴Number and percent with the given score ⁵GCT: Germ cell tumors ⁶StromaT: Granular cell tumors ⁷Krukenberg T: Krukenberg tumors ⁸Mets. AdCa:Metastatic adenocarcinoma ⁹Mucous P AdCa: Mucous papillaryadenocarcinoma ¹⁰CCC: Clear cell carcinoma

Expression of HERV-K env SU protein increased in a stepwise fashion fromgrade I (33%) to grade II (38%) to high-grade (47%) serous papillaryadenocarcinoma (FIG. 9A) for 40 serous papillary adenocarcinoma tissuesobtained from the TMA1 tissue microarray. Microarray TMA2 containednormal, mucinous cyst, LMP, LG, and HG carcinomas, and this array wasused for analysis of progression of ovarian cancer (FIG. 9B). LMPserous, LG serous, and LG endometrial tumors showed higher levels ofexpression compared to normal ovaries (p<0.001). HG serous andendometrial tumors showed great variability in protein expression with amedian expression slightly lower than normal ovaries. Furthermore,tissue microarray TMA3, containing 484 cases of various ovarian cancertissues with clinical follow-up information, was used to assess whetheractivation of HERV-K env surface protein correlated with clinical orhistological characteristics, or with prognostic factors associated withthe patients. The parameters evaluated included patient demographics,hormone receptor status, tumor type, tumor stage, and survival. Astatistically significant increase in HERV-K expression was observed forhistotypes with a diagnosis of ovarian cancer (Table 4). There were nosignificant increase in tumor grade, stage, patient age, and level ofcytoreduction achieved (data not shown). Clear cell and mucinouscarcinomas showed lower levels of expression compared to other ovariancancers. Patients with low expression of HERV-K env SU protein had thehighest survival rate. However, patients with mid-level expression ofHERV-K env SU protein had lower survival rate than patients with highexpression (data not shown).

TABLE 4 Ovarian cancer tissue microarray: HERV-K envelope surfaceprotein expression and clinicopathological characteristics. Histotype 01 2 + 3 Total Endometrioid adenocarcinoma 6 (12.8%) 11 (23.4%) 30(63.8%) 47 Serous carcinoma 38 (10.4%) 109 (29.8%) 219 (59.8%) 366Malignant mixed müllerian tumor 1 (5.9%) 7 (41.2%) 9 (52.9%) 17 Clearcell carcinoma 5 (26.3%) 8 (42.1%) 6 (31.6%) 19 Poorly differentiatedcarcinoma 4 (25%) 2 (12.5%) 10 (62.5%) 16 Mucinous adenocarcinoma 4(40%) 4 (40%) 2 (20%) 10 Transitional cell carcinoma 3 (33.3%) 6 (66.7%)9 p value*  0.01 Tumor grade 1 4 (18.2%) 10 (45.5%) 8 (36.4%) 22 2 4(16.7%) 5 (20.8%) 15 (62.5%) 24 3 49 (11.5%) 129 (30.2%) 249 (58.3%) 427missing 1 (9.1%) 10 (90.9%) 11 p value  0.24 Stage Stage 1 6 (15%) 12(30%) 22 (55%) 40 Stage2 3 (8.3%) 11 (30.6%) 22 (61.1%) 36 Stage3 36(12.9%) 77 (27.5%) 167 (59.6%) 280 Stage 4 11 (11.8%) 34 (36.6%) 48(51.6%) 93 missing 2 (5.7%) 10 (28.6%) 23 (65.7%) 35 p value 0.7 Age <5520 (12.2%) 44 (26.8%) 100 (61%) 164 >55 34 (12.1%) 89 (31.6%) 159(56.4%) 282 missing 4 (10.5%) 11 (28.9%) 23 (60.5%) 38 p value 0.5 Levelof cytoreduction achieved suboptimal 25 (12.1%) 65 (31.4%) 117 (56.5%)207 optimal 27 (12.4%) 63 (28.9%) 128 (58.7%) 218 unknown 2 (66.7%) 1(33.3%) 3 missing 6 (10.7%) 14 (25%) 36 (64.3%) 56 p value 0.8 *P valuescalculated using chi-square test of independence.

Example 14 Detection of Anti-HERV Antibodies in Sera of Ovarian CancerPatients

To determine whether HERV proteins expressed in ovarian cancer tissuesare immunogenic in ovarian cancer patients, we assayed for anti-HERVantibodies in sera from patients with various cancers including ovariancancer. ELISA analysis was employed to determine the binding affinityand specificity of the sera obtained from ovarian cancer patients (n=60)and normal female controls (n=50). The cutoff value is 0.5 for OD at 405nm. Approximately 55% of the 60 ovarian cancer patient samples werepositive for antibodies against HERV-K surface protein, 40% werepositive for antibodies against HERV-E surface protein, 55% werepositive for antibodies against HERV-K gag protein, 50% were positivefor antibodies against an env protein splice variant, and 30% werepositive for antibodies against ERV3 env protein. ELISA results obtainedfrom 20 ovarian cancer patients and 20 normal female controls are shownin FIGS. 10A and B. The presence of anti-HERV env protein antibodiesprovides indirect evidence of the presence of HERV proteins in humanovarian cancer. Further, the occurrence of these antibodies in thecirculation of cancer patients but not normal subjects indicates thatHERV may be an unrecognized tumor-associated antigen in ovarian cancer.

Materials and Methods for Examples 10-14

Cells and tissues. The human ovarian cancer cell lines PA1 and SKOV3were obtained from the American Type Culture Collection (ATCC;Rockville, Md.) and were cultured in the media recommended by themanufacturers. The human ovarian surface epithelial cancer cell linesOVCA 430, OVCA 433, OVCA 420, OVCAR3, DOV 13 and OVCA 429, and thenormal human ovarian epithelial cell lines NOE 114, NOE 116, NOE 113,and NOE 119 were gifts from Dr. Robert C. Bast Jr., University of TexasM. D. Anderson Cancer Center. The normal human ovarian epithelial celllines T29, T72, and T80 were generated from human ovarian surfaceepithelial cells that had previously been transfected with the SV40early region expressing large T and small t antigens, and which wereinfected subsequently with a retrovirus containing a full-length hTERTcDNA, as described in Liu J, et al. Cancer Res 64:1655-1663 (2004). Thehuman ovarian cancer cell lines were maintained in minimal essentialmedium supplemented with 10% Bovine Growth Serum (BGS; HyClone),penicillin and streptomycin, glutamine, non-essential amino acids andsodium pyruvate. For hormone stimulation, cells were treated withβ-estradiol on day 1 and progesterone on day 2 as described inRininsland F H, et al., J Immunol Methods 240(1-2):143-55, 2000. Thenormal human surface ovarian epithelial cell lines were cultured in a1:1 mixture of MCDB 105 medium (Sigma) and Medium 199 (LifeTechnologies, Inc.) supplemented with 10 ng/ml epidermal growth factor(Sigma), 15% BGS, L-glutamine, penicillin and streptomycin. Tissuesamples were snap-frozen and stored at −70° C. until RNA isolation. Forin situ hybridization or immunohistochemistry, formalin-fixed,paraffin-embedded tissues were used.

PCR primers. Oligonucleotide primers derived from the sequences encodingthe env surface proteins of HERV-K, ERV3 and HERV-E were used to amplifycDNA prepared from human ovarian tissues and cell lines as described inWang-Johanning F, et al. Clin Cancer Res 7:1553-1560, 2001. The 5′ senseprimer of the HERV-K env gene has between one and four base-pair (bp)mismatches with most type 2 HERV-K env genes. In this study, a senseprimer (nucleotide [nt] 6674-6698; Accession number: AF074086 (Mayer J,et al, Nat Genet 21:257-258, 1999)) specific for type 2 HERV-K env geneswas also used to detect type 2 HERV-K env mRNA transcripts, as describedin Wang-Johanning F. Oncogene 22:1528-1535, 2003. Previously-describedprimer pairs were used to amplify env reading frame transcripts thatinclude np9. Armbruester V, et al. N. Clin Cancer Res 8:1800-1807, 2002.

RT-PCR. RNA was prepared and treated with DNase as described in anearlier study. Wang-Johanning F, et al. Clin Cancer Res 7:1553-1560,2001. Briefly, isolated total RNA was incubated at 65° C. for 10 minutesfollowed by incubation on ice for 2 minutes prior to reversetranscription. Reverse transcription was carried out for 1 h at 37° C.using cDNA synthesis beads (Amersham Pharmacia Biotech Inc., Piscataway,N.J.) as per the manufacturer's instructions. The reverse transcribedsamples were amplified in a volume of 50 μl using the HERV env sense andantisense primer pairs described in Wang-Johanning F, et al. Clin CancerRes 7:1553-1560, 2001. The same reverse-transcribed RNA sample wasanalyzed using primers that recognize human β-actin to confirmequivalent loading. One microgram of RNA from the same sample withoutreverse transcriptase addition was amplified in parallel to ensure thatno genomic DNA was present in the samples.

Real-time RT-PCR. One-step RT-PCR was performed using an ABI PRISM7900HT sequence detector to quantitate the expression of HERV-K env genein various ovarian specimens. The optimized concentrations of HERV-Kprimers and probes were determined and used for real-time RT-PCR asdescribed in Wang-Johanning F. Oncogene 22:1528-1535, 2003. Homo sapiensribosomal protein S9 (GenBank accession number XM 008957.2) was used asan endogenous control. Wang-Johanning F, et al. Cancer 94:2199-2210,2002. Briefly, the amplification reactions were performed in 25 μl finalvolume containing 1× TaqMan buffer (Perkin-Elmer Applied Biosystems,Foster City, Calif.) plus dNTPs (0.3 mM each), 0.625 units of AmpliTaqGold, RNase inhibitor (5 units), 2% glycerol, and 0.625 units of MuLVreverse transcriptase. All RT-PCR reactions were performed in opticalreaction tubes (Perkin-Elmer) designed for the ABI PRISM 7900HT sequencedetector system. Reverse transcription and thermal cycling conditionswere 30 min at 48° C. followed by 10 min at 95° C., and 40 cycles of 15s at 95° C. and 1 min at 60° C. PCR premixes containing all reagentsexcept for total RNA were used as no a template control. Linearextrapolation of the cycle threshold (CT) values of HERV-K were obtainedfrom ovarian specimens, and were then divided by the relative amounts ofS9, which were quantitated by linear extrapolation from the CT values ofthe same unknown samples.

Synthesis of HERV env fusion proteins and production of anti-HERV envprotein antibodies. HERV cDNAs obtained from cancer tissues were clonedinto the corresponding enzyme-digested QIA expression vector (pQE30;Qiagen Inc.), which contains a 6-His tag at the N-terminus, or pGEXvector (4T1; Amersham Pharmacia), which contains glutathioneS-transferase (GST). After screening for protein production on asmall-scale, the HERV env-positive colonies were further identified byrestriction enzyme analysis and characterized by sequencing usingvector-specific primers to confirm that the clones produced the desiredHERV env proteins. The HERV env-positive colonies were induced withisopropyl-B-D-thiogalactopyranoside and purified by affinitychromatography using Ni-nitrilotriacetic acid agarose (Qiagen) for thepQE vector, or Glutathione Sepharose 4B (Amersham Pharmacia) for thepGEX vector. These purified HERV env fusion proteins were used toimmunize rabbits for polyclonal or mice for production of anti HERV envprotein monoclonal antibodies using standard techniques. The antibodieswere further purified using Protein G Sepharose 4 Fast Flow (AmershamPharmacia) and tested for specificity and sensitivity against variousHERV env proteins by ELISA and/or immunoblot analysis.

Flow cytometry. Cultured cells were incubated with anti-HERV-K antibody(5691; 1:200 dilution) at 4° C. for 30 min, followed by anti-rabbitIgG-FITC secondary mAb (1:1000 dilution) at 4° C. for 15 min. Forstaining in permeabilized conditions, cells were treated with 0.1%Triton X-100 in PBS, then incubated with primary antibody, followed bysecondary antibody. After washing with PBS, the cells were fixed with 3%paraformaldehyde in PBS. Samples were analyzed on a BD FACSCalibur™system (BD Biosciences). For controls, samples were incubated withanti-rabbit IgG-FITC secondary mAb.

Immunohistochemistry for ovarian tissue slides. Immunohistochemistry wasperformed on a range of human ovarian tumor and non-tumor tissues usingpre-immune serum and various anti-HERV antibodies. Paraffin-embeddedovarian tissue specimens were cut into serial 5 μm sections, melted,deparaffinized in xylene, rehydrated in ethanol and then fixed in 4%paraformaldehyde. The slices were incubated with horse sera, anti-HERV-Kenv polyclonal antibody (1:200 dilution), pre-immune serum (as anegative control; 1:200 dilution) or NCL-5D3 monoclonal antibody forcytokeratin 8/18 (Vector Laboratories Inc., Burlingame, Calif.) as apositive control to identify glandular epithelium or adenocarcinomas(1:40 dilution). This was followed by incubation with anti-rabbit IgGbiotin conjugate antibody (1:1,000 dilution) or anti-mouse IgG biotinconjugate antibody, and finally with ABC (ABC kit, Vector) as describedby the manufacturer. Diaminobenzidine (Vector Laboratories) substratewas used for color development. Slices were then counterstained withhematoxylin.

Tissue microarray (TMA) slides. Multiple tissue microarray slide TMA1,containing 72 ovarian tissues from patients with various ovariandiseases, was obtained from US Biomax, Inc (Catalog # CC11-01-002;Rockville, Md.). Slide TMA2 contains 85 ovarian tissues that includednormal, mucinous cyst, low malignant potential, low-grade, andhigh-grade carcinomas obtained from The University of Texas M. D.Anderson Cancer Center Department of Pathology. TMA3 contain 484 casesof various ovarian cancer tissues obtained from University of Texas M.D. Anderson Cancer Center, with clinical follow-up information.

Immunohistochemistry for multiple tissue microarray slides.Immunohistochemistry was performed on tissue microarray slides using aDAKO autostainer universal staining system (Model: LV-1). The DAKOAutostainer System is an automated slide processing system compatiblewith currently available reagents for the staining of paraffin-embeddedand frozen tissue sections. These multiple tissue microarray slidesprovided us with a means to compare the expression of HERV-K env SUprotein in multiple tissues under identical conditions of staining. Theprotocol has been programmed into the system, and the slices wereincubated with 3% H₂O₂ (5 min), horse sera (10 min), and antibodies(1:750 dilution for anti-HERV-K antibodies and 1:100 dilution forNCL-5D3) (30 min). This was followed by incubation with anti-rabbit oranti-mouse IgG HRP conjugate antibody (DAKO) (15 min), incubation withdiaminobenzidine (5 min) for color development, and counterstaining withhematoxylin (5 min).

ELISA. ELISA assays were used to detect anti-HERV antibody in humansera, and were carried out as described in Wang-Johanning F, et al.Cancer Res 58: 1893-1900, 1998. Briefly, a 96-well ELISA plate wascoated with various HERV env fusion proteins (10 μg per ml, 100 μl perwell) in PBS and incubated overnight at 4° C. The plate was then blockedfor 1 h with 5% nonfat dry milk (Sigma) and 3% BSA at room temperature.Human sera (1:200 dilution with PBS) were added to the coated wells, andthe plate was incubated overnight at 4° C. After washing 6 times withPBS-T (0.5 ml of Tween 20 in 1000 ml of PBS), 100 μl of HRP-conjugatedanti-human IgG antibody (1:2000 dilution, Sigma) was added to each wellof the plate to detect the serum antibody, followed by incubation for 1h at room temperature. The plate was washed again with PBS-T, and colorwas developed using ABTS (Sigma). After 10 min, absorbances of the platewells were measured on a microplate reader at 405 nm. All ELISA analyseswere performed at least three times for each serum sample. The means ofthe threshold values (Wang-Johanning F, et al. Cancer Res 58: 1893-1900,1998) were used for the final analysis.

Example 15 Specificity and Sensitivity of Anti-HERV-K Antibodies

Monoclonal antibodies against HERV were also produced in our laboratory,including anti-HERV-K env, and anti-HERV-E env mAbs. Anti-HERV-K orHERV-E positive clones were used to test for their specificity orsensitivity against HERV-K env (FIG. 11A) or HERV-E env (FIG. 11B)proteins by ELISA analysis. Several other anti-HERV protein monoclonalantibodies, including anti-HERV-E and anti-ERV3 antibodies, have beenproduced, including anti-HERV-K spliced-env antibodies, anti-HERV-K gagantibody, anti-ERV3 env antibody, and anti-HERV-E env antibody.

Example 16 Investigation of the In Vitro and In Vivo Antitumor Effect ofAnti-HERV-K Antibodies

Anti-HERV-K antibody has been observed to inhibit proliferation ofbreast cancer cells (MCF-7) and ovarian cancer cells (DOV13), but notnormal or benign breast (MCF-10A and MCF-10AT) or ovarian (T 80) celllines, as shown in FIG. 12.

The anti HERV-K antibody alone induces MCF-7 cancer cells (25%) andDOV13 (20%) to undergo apoptosis. Testing revealed that these antibodiesare pure and have high specificity for their targets.

The antitumor effect of anti-HERV-K antibody has been demonstrated inmice bearing murine mammary tumors expressing HERV-K env protein, where60% of mice treated with antibody remained tumor free. No antitumoreffect was detected in mice treated with control antibody or in micebearing HERV-K negative tumors.

Both B6D and B6DK cells (5×10⁶) were injected s.c. into the right flankof mice (H-2^(b)), and average tumor sizes in mice were compared. Tumorssizes were 2.42-fold greater in mice with B6DK cells than their parentcells (at 40 days post-injection). However, mice that were immunizedwith HERV-K env surface proteins were protected from subsequent tumorchallenge. No change in tumor growth rate was detected in mice bearingB6D cells, which are HERV-K negative parent cells. Furthermore, bonemarrow-derived DCs were administrated as vaccines to improve theantitumor activity. DCs were pulsed with HERV-K env surface protein,control protein (such as HPV16E6 protein in the same expression vector;pQE30), HERV-K env cRNAs constructed by in vitro transcription, orcontrol cRNA (such as HPV16E6 cRNA in the same vector; pcDNA3). Resultsof a representative study are depicted in FIG. 14A. In addition, theantitumor effect was observed in the mice bearing B6DK mammary tumorexpressing HERV-K env protein treated with DC pulsed with KcRNA andpeptides Kp201 and Kp640. No protection was observed in animals treatedwith nonpulsed DCs (FIG. 14B). p1028 peptide was used as positivecontrol.

Example 17 Construction of a Single-Chain Anti-HERV-K Antibody(Designated HERV-K sFv) and Fusion to the Recombinant Toxin Gelonin(rGel)

Cloning of the VH and VL domains of anti-HERV-K antibody from micehybridomas (monoclonal antibodies) or spleen cells. mRNA from murinehybridoma 4D1 expressing anti-HERV-K antibody (IgG2A) or spleen cellsobtained from Balb/c mice has been isolated and reverse-transcribed tocDNA, and spleen cells obtained from HLA-A2 transgenic mice will beisolated and reverse-transcribed to cDNA. Amplification of antibodylight- and heavy-chain variable regions was carried out using the Vheavy chain or light chain primers as described in Wang-Johanning F, etal. Cancer Res 58: 1893-1900, 1998. DNA amplified using this procedurewas then cloned into the Invitrogen T/A cloning vector pCR II withoutfurther purification, transformed into Escherichia coli XL1-Blue, andidentified using blue-white screening procedures. Positive clones (fiveeach from the heavy- and light-chain libraries) were sequenced using theT-7 and SP6 promoter primers (see sequence in FIG. 15) (SEQ ID. NO:11),and antibody domains will be identified by homology to otherimmunoglobulin sequences.

Construction of genes encoding the single-chain antibody HER V-K sFv andthe immunotoxin HER V-K sFv-rGel. A two-step splice-overlap extensionPCR method (Rosenblum, M. G. Cancer Res 63, 3995-4002, 2003) will beused to construct the single-chain antibody HERV-K sFv using light- andheavy-chain DNA clones as templates. The clones will then be fusedtogether using the splice-overlap extension PCR method with gelonin DNAas templates. PCR products will be purified and digested with BamHI andHindIII as described previously, and cloned into vector pQE-30.Sequenced DNA clones will be subsequently transformed into E. colistrain M15 obtained from Qiagen for expression of the fusion toxin. Thecolony-blot procedure will be used for identification of clonesexpressing anti-HERV-K antibody. After the positive clones expressinganti-HERV-K are identified, the clones will be sequenced to confirm thatthe sequence is correct.

Protein expression in E. coli. The positive bacterial cultures will beincubated at 37° C. in 2×LB growth medium with strong antibioticselection (200 μg/ml ampicillin, and 15 μg/ml kanamycin) and grown untilearly log phase (A600 nm=0.4-0.8). The cultures will then be induced at37° C. by the addition of 0.1 mM IPTG for 4 h. Induced bacterialcultures will be centrifuged and purified by affinity chromatography asdescribed previously (F Wang-Johanning, 2001, Clinical Cancer Research).

Internalization and immunofluorescence staining. Antigen-positive (MCF-7or DOV13) cells will be added to polylysine-coated 16-well chamberslides (Nunc) at 104 cells/chamber and incubated at 37° C. overnightunder 5% CO₂ atmosphere. Cells will be treated with 50 μg/ml HERV-KsFv-rGel fusion construct for various time intervals. Cells will bewashed briefly with PBS, and proteins bound to the cell surface will bestripped by 10-min incubation with glycine buffer (500 mM NaCl and 0.1 Mglycine (pH 2.5)), neutralized for 5 min with 0.5 M Tris (pH 7.4),washed briefly with PBS, and then fixed in 3.7% formaldehyde (Sigma) for15 min at room temperature, followed by a brief rinse with PBS. Cellswill then be permeabilized for 10 min in PBS containing 0.2% TritonX-100, washed three times with PBS, and incubated with PBS containing 3%BSA for 1 h at room temperature. After a brief wash with PBS, cells willbe incubated with rabbit anti-rGel polyclonal antibodies diluted 1:500in PBS containing 0.1% Tween 20 and 0.2% BSA for 1 h at roomtemperature. Cells will be washed three times in PBS containing 0.1%Tween 20 for 10 min and blocked for 1 h at room temperature with PBScontaining 3% BSA, followed by a 1:100 dilution of FITC-coupledantirabbit IgG (Sigma) containing 2.5 μg/ml of propidium iodide (PI).Control cells will be incubated only with the secondary FITC-coupledantirabbit IgG (1:100) plus 2.5 μg/ml of PI. After three final washeswith PBS containing 0.1% Tween 20, cells will be washed once with PBSfor 10 min and mounted in DABCO mounting medium containing 1 μg/ml ofPI. Slides will then be analyzed with a fluorescence microscope.

In vitro cytotoxicity assay. Samples will be assayed using a standard72-h cell proliferation assay with log-phase (5000 cells/well)antigen-positive MCF-7 and DOV13 cells, and antigen-negative MCF-10A andT80 cell monolayers, using crystal violet staining procedures asdescribed in Rosenblum, M. G. Cancer Res 63, 3995-4002, 2003.

TUNEL assay. Log-phase MCF-7 cells will be plated into 16-well chamberslides (10,000 cells/well) and incubated overnight at 37° C. in a 5% CO2atmosphere. Cells will be treated with the fusion protein HERV-KsFv-rGel or rGel at a final concentration of 87 nM for different timeperiods (24 and 48 h) and washed briefly with PBS. Cells will be fixedwith 3.7% formaldehyde at room temperature for 20 min, rinsed with PBS,then permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate on icefor 2 min, and washed twice with PBS. Cells will be incubated with TUNELreaction mixture at 37° C. for 60 min, followed by incubation withConcerter-AP at 37° C. for 30 min, and finally reacted with Fast Redsubstrate solution at room temperature for 10 min. After a final washstep, the slides will be mounted in mounting medium and analyzed under alight microscope. Positive controls will be included in eachexperimental set up. Fixed and permeabilized cells will be incubatedwith 1 mg/ml DNase I for 10 min at 37° C. to induce DNA strand breaks.

In vivo cytotoxicity studies. Athymic (nude) female mice or HLA-A2transgenic female mice (4-6 weeks old) will be divided into groups of 5mice/cage. Log-phase MCF-7 or DOV13 human cancer epithelial cells (5×10⁶cells/mouse) will be injected s.c. in the right flank, and tumors willbe allowed to establish. Ovarian cancer cells expressing greenfluorescent protein as a result of transfection with a PG13-GFPexpression vector will also be injected by an i.p. route, and thesetumors can be detected using a fluorescence flashlight. Once tumors aremeasurable (˜30-50 mm²), animals will be treated (i.v. via tail vein)with either saline (control) or various concentrations of the HERVsFv-rGel fusion toxin for 4 consecutive days. Animals will be monitored,and tumors will be measured for an additional 30 days.

Example 18 Determination of Specificity and Sensitivity of Anti-HERV-KAntibody

Bacterial colonies positive for HERV-K env expression, characterized bysequencing, were induced with isopropyl-B-D-thiogalactopyranoside andpurified by affinity chromatography using Ni-NTA Resin (Qiagen Inc.) orGlutathione Sepharose 4B by AKTAprime plus (GE Healthcare Bio-SciencesCorp). The purified HERV-K env fusion proteins were used for productionof IVS cells, or to immunize rabbits or mice for the production ofpolyclonal or monoclonal anti-HERV-K env antibodies, respectively, usingstandard techniques. The antibodies were further purified and tested forspecificity and sensitivity.

HERV-K env surface (K-SU) protein was cloned into two expression vectors(pQE30 and PGEX4T1) to generate HERV-K recombinant fusion proteinsK10Q18 and K10G17, respectively, as described previously(Wang-Johanning, F., A. R. Frost, G. L. Johanning, M. B. Khazaeli, A. F.LoBuglio, D. R. Shaw, and T. V. Strong. 2001. Expression of humanendogenous retrovirus k envelope transcripts in human breast cancer.Clin Cancer Res 7:1553-1560; Wang-Johanning, F., J. Liu, K. Rycaj, M.Huang, K. Tsai, D. G. Rosen, D. T. Chen, D. W. Lu, K. F. Barnhart, andG. L. Johanning. 2006. Expression of multiple human endogenousretrovirus surface envelope proteins in ovarian cancer. Int J. Cancer.)K10Q18 was used to immunize animals to generate antibodies and K10G17was used to screen for specificity and sensitivity of these antibodies.Several polyclonal and monoclonal anti-HERV-K antibodies were producedby standard methods. The positive clones were tested for theirspecificity and sensitivity against K-SU protein by ELISA and Westernblot. Sample ELISA and Western blot results are shown, respectively, inFIG. 11A and FIG. 11C (top panel). Five hybridoma clones (4E11, 4D1,4E6, 6E11, and 6H5) had higher sensitivity against K-SU protein thanagainst HERV-E env surface protein, another HERV family member (clonedinto pQE30 vector). Also, HERV-K fusion protein K10G17 was detected bytwo anti-HERV-K monoclonal clones, 4D1 and 6H5, which were generated byimmunization with K10Q18 fusion protein. Anti-GST mAb was the positivecontrol (data not shown).

Detection of anti-HERV antibodies in BC patients. The reactivity ofanti-HERV-K antibodies obtained from BC patients and healthy femalecontrols toward recombinant K-SU protein was determined by Western blot.Three patients with invasive ducal carcinoma, but not a healthy femaledonor, had anti-HERV-K serum antibodies which detected K10G17 protein(FIG. 1 IC, bottom panel), just as did monoclonal antibodies 6H5 and 4D1(FIG. 11C, top panel). The sensitivity and specificity of antibodies insera or plasma of BC patients toward K-SU was determined by ELISA. ELISAresults for one series of serum dilutions (FIG. 11D) revealed higherK-SU antibody titers (p<0.001 to 0.005; Student's t test) in BC patientsthan in control subjects. The cut-off value was 0.5 for optical densityat 405 nm. Approximately 50% of the BC patient samples (n=48) werepositive for antibodies against K-SU protein, 15% had antibodies againstHERV-K gag protein, and 35% had antibodies against type 2 HERV-K envprotein with a 292 bp insert. In contrast, anti-HERV-K antibodies werenot detected in control samples (N=50). The presence of anti-HERV-K envprotein antibodies provides indirect evidence of the presence of HERV-Kenv proteins in human BC, which indicates that there was a humoralresponse to HERV-K.

HERV-K-specific CD4⁺ T cell responses. Dendritic cells (DC) weregenerated from PBMC cultured in medium containing the cytokines GM-CSFand IL-4. Immature DC were exposed to TNF-α overnight for maturation,with or without prior pulsing with HERV-K proteins.Fluorescence-activated cell-sorting (FACS) analysis revealed thatHERV-K-pulsed mature DC had enhanced CD83 expression compared withimmature DC and mature DC treated with TNF-α only. Expression ofCD83⁺/CD209⁺ and CD83⁺/CD86⁺ was also higher in HERV-K-pulsed mature DCthan in immature DC (data not shown).

PBMC obtained from BC patients (Table 7) or normal matched age femaledonors were stimulated with autologous mature DC pulsed with HERV-K envantigen for 1 week and assessed for proliferation using a ³H-thymdineincorporation assay. T cell proliferation was detected in one week IVSobtained from BC patients but not normal donors. Similar results wereobtained when DC were pulsed with either K-SU protein (FIG. 17A) orHERV-K env surface RNA produced by in vitro transcription (IVT) usingHERV-K env surface cDNA as a template (FIG. 17B). As shown in thescatter plots in FIG. 17C, the fold increase in HERV-K-specific T cellproliferation relative to autologous PBMC was significantly higher inIVS cells from cancer patients (N=16) than in those from healthy controlsubjects (N=18; p=0.023; Student's t test). In addition, when total PBMCor isolated CD3⁺ cells obtained from healthy donors (N=7) werestimulated with HERV-K or HERV-E env surface protein (another HERVfamily), neither HERV-K nor HERV-E env surface protein stimulated humanT cell proliferation to a similar extent as the superantigenStaphylococcal enterotoxin A (data not shown). These results indicatethat T-cell proliferative responses against HERV-K are found only in BCpatients and not in individuals without cancer.

TABLE 7 Patient characteristics Patient sample Age Lymph node Date ofdiagnosis number (years) Diagnosis status (month/year) C1 51 IDC¹12/1992  C2 59 IDC 4/2004 C3 40 DCIS² + ILC³ Negative 6/2004 C4 54DCIS + IDC 3/2003 C5 41 IDC stage 3 9/2004 C6 41 IDC Negative 4/2004 C749 IDC Negative 1/2000 C8 55 DCIS Negative 6/2005 C9 32 IDC Negative10/2003  C10 55 IDC + colon C⁴ Positive 6/2005 C11 38 IDC Positive6/2005 C12 49 DCIS Negative 6/2005 C13 42 DCIS Negative 11/2005  C14 79IDC Negative 1994 BC 687150 64 IDC + SCC⁵ 7/2006 BC 691965 66 IDC 9/2006BC 691271 49 IDC 8/2006 BC 684700 43 DCIS 8/2006 BC 695606 55 DCIS +IDC + ILC 10/2006  ¹IDC, infiltrating ductal carcinoma, ²DCIS, ductalcarcinoma in situ, ³ILC, invasive lobular carcinoma, ⁴colon C, coloncancer, ⁵SCC, squamous cell carcinoma

HERV-K specific CD8⁺ T cell responses. We then determined HERV-Kspecific Granzyme B (GrB) or IFN-γ release using an ELISPOT assay or⁵¹Cr CTL assays. Results from a representative experiment are shown inFIG. 18A. HERV-K-specific GrB (top panel) or IFN-γ spot numbers (bottompanel) detected by ELISPOT after 3-week IVS (PBMC were stimulated twicewith DC pulsed with K-SU) were significantly higher in cancer patientsthan in healthy controls. Similar results were obtained with PBMCstimulated in 1-week IVS assays (data not shown). Very few or no GrBspots were detected from PBMC stimulated with unpulsed DC obtained fromhealthy donors or cancer patients. A significantly greater number of GrBspots were produced in BC patients (N=13) than in normal female donors(N=16), regardless of whether they were subjected to 1 week IVS or 3week IVS (FIG. 18B).

⁵¹Cr-release CTL assays were employed to compare HERV-K specific CD8⁺ Tcell responses between BC patients and normal donors. An example of aCTL assay after 1 wk IVS is shown in FIG. 18C. IVS from four normaldonors stimulated with HERV-K-expressing DC had lower antigen-specificCTL activity than IVS from four cancer patients. Higher cytotoxicactivity in BC patients than in normal donors against K-SU protein(DC+Kpro) or RNA (DC+KRNA) was observed, with lesser cytotoxic activityagainst HPV16 E6 protein (DC+E6pro) or RNA (DC+E6RNA which obtained byIVT using E6 DNA a template; data not shown). Other human proteinsproduced in the pQE30 expression vector, including LMP2A and HPV16 E7,had lesser cytotoxic activity than K-SU protein (data not shown). PBMCfrom normal donors stimulated with HERV-K-expressing DC in IVS assaysdid not exhibit any antigen-specific CTL activity (FIG. 18C).

Anti-HERV-K antibody inhibited BC cell proliferation and induced BCcells to undergo apoptosis. The antitumor effect of α-K antibodies in BCcells was determined by an MTS cell proliferation assay. Antibodies wereable to inhibit BC, but not normal or benign breast cell proliferation.Anti-HERV-K pAb 5693 inhibited proliferation of MCF-7 BC cells, but notbenign MCF-10A breast cells (FIG. 19A). The cytotoxicity of α-K mAb 6H5toward BC cell lines was compared. The IC₅₀ of 6H5 mAb for MCF-7 andMDA-MB-231 was 51.7 nM and 6.6 nM, respectively, and 6H5 showed nocytotoxicity toward MCF-10A cells (FIG. 12). Furthermore, weinvestigated whether α-K mAbs induce apoptosis in BC cells. Breast cellstreated with α-K mAb (6H5; 0, 1.25, 2.5, 5, 10 μg/ml) for 24 hr weresubjected to FACS analysis of Annexin V-APC and 7AAD-PEcy7 expression,to assay for apoptosis. 6H5 induced apoptosis in a dose-response fashionin MCF-7 cells, and the response in MCF-7 cells was greater than inMCF-10AT; MCF-10A did not undergo apoptosis in response to 6H5 (FIG.19B). Apoptosis in these breast cell lines is summarized in FIG. 19C.Several α-K mAb clones were able to induce apoptosis in T47D and NIHMCF-7 BC cells, but not in MCF-10A or MCF-10AT breast cell lines.Anti-HERV-K mAb induced apoptosis to a greater extent in BC cells thanin premalignant breast cells, but had no effect on apoptosis in benignbreast cells.

Example 19 Adoptive T Cell Therapy Inhibits Breast Tumor Growth in Mice

We evaluated whether a combination of HERV-K specific CTLs and α-Kantisera function synergistically in tumor regression in vivo byevaluating influences of immunization on the course of tumor appearance.We first induced anti-K-SU immune responses in HLA-A2 mice (A2 mice),and then used the CTLs and antisera from these mice for adoptive T celltherapy in SCID mice. These experiments provide essential information asto whether HERV-K proteins can serve as tumor targets for development ofa vaccine against breast cancer in vivo. The tumor sizes were reduced by61% in SCID mice treated with CD90⁺ T cells from A2 mice, plus antiserafrom A2 mice (174 mm³) (P<0.05) compared with untreated controls (452mm³). In addition, tumor sizes were reduced by 80% in the 6H5 mAb group(93 mm³) (P<0.05) relative to controls. The tumor sizes showed nosignificant difference relative to controls in the mice treated withantisera alone (199 mm³) or CD90⁺ T cells alone (293 mm³). (FIG. 20A.)FIG. 20B illustrates tumor formation in mice innoculated with MCF-7cells on day 0 and treated with saline or 6H5 on days 4, 6, and 8(arrows; 200 ug per mice). Mice treated with saline were used ascontrol.

Example 20 Studies with Anti-HERV-K Antibody, 6H5

Many studies were performed on various breast and ovarian cell linesusing anti-HERV-K Antibody, 6H5. ELISA and Western Blot were performedon the samples to determine monoclonal antibody specificity and proteinexpression, as described previously. Immunofluorescence staining,confocal microscopy, flow cytometry, and dry cell ELISA were performedto determine the surface expression of the viral protein.Internalization assays were performed to detect toxin entry into targetcells; immunohistochemistry was performed to protein expression in tumorbiopsies. Cytoxicity analysis was conducted to determine IC₅₀ of mAb ormAb-rGel in various cells. Nude or SCID mice were treated with mAB totest for protection against tumor growth. Western blot of various breastcell lines and an ovarian cell line using 6H5 mAb to detect HERV-K envexpression can be seen in FIG. 16E. FIGS. 16E and 16F show thatanti-HERV-K antibodies were detected in sera obtained from breast cancerpatients. Western blot of various ovarian cell lines using 6H5 mAb todetect expression of HERV-K env protein can be seen in FIG. 21.

Surface and cytoplasmic expression of HERV-K env protein in ovariancancer cells was detected by confocal microscopy using 6H5 mAb. rGel wasdelivered into DOV13 cells by 6H5, and was detected by anti-rGel Ab.Coomasie blue stain of 6H5 mAb and 6H5-rGel conjugate can be seen inFIG. 29. The results of this study are illustrated in FIG. 22. Surfaceand cytoplasmic expression of HERV-K env protein in breast cell linesdetected by confocal microscopy using 6H5 mAb are illustrated in FIGS.23 and 24. rGel was delivered into MCF-7 cells by 6H5, and was detectedwith anti-rGel Ab.

Surface expression of HERV-K env protein in ovarian cell lines wasquantitated by ELISA using 6H5 mAb. Murine IgG was used as a negativecontrol. These results can be seen in FIG. 25. Quantitation of surfaceexpression of HERV-K env protein in ovarian cell lines by FACS using 6H5mAb can be seen in FIG. 26. Murine IgG was used as a negative control.

The ability of 6H5 to induce ovarian cells to undergo apoptosis wasassessed, as compared to cells that were not treated with antibody. FIG.27 shows the results of this study. The results indicated that theantibody against HERV-K is able to induce cancer cells to undergoapoptosis. Apoptosis in these ovarian cell lines is summarized in FIG.28. The results of comparative cytotoxicity studies with breast celllines and ovarian cell lines can be seen in FIGS. 30 and 31.

The results of this study indicate that 6H5 mAb is able to inhibitcancer cell proliferation by the MTT assay. 6H5 mAb is also able toinduce cancer cells to undergo apoptosis. 6H5 mAb-rGel is cytotoxic tocancer cells, but not normal cells. Expression of HERV-K env protein wasobserved only in cancer cells of biopsies by immunohistochemistry using6H5. HERV-K envelope protein is a tumor specific antigen, and can beused for detection, diagnosis, and as a target for immunotherapy. Bothsurface and cytoplasmic expression of HERV-K env protein is observed oncancer cells only, which suggests that HERV-K can stimulate both T celland B cell responses. 6H5, a mAb against HERV-K env protein, is able totreat HERV-K positive cancers. 6H5 is useful as an immunotoxin, and canalso be used for radiotherapy and as an imaging agent.

Methods for Examples 15-20.

ELISA and Western blot assays. ELISA and Western blot assays wereperformed as described previously (Wang-Johanning, F., J. Liu, K. Rycaj,M. Huang, K. Tsai, D. G. Rosen, D. T. Chen, D. W. Lu, K. F. Barnhart,and G. L. Johanning. 2006. Expression of multiple human endogenousretrovirus surface envelope proteins in ovarian cancer. Int J Cancer;Wang-Johanning, F., G. Y. Gillespie, J. Grim, C. Rancourt, R. D.Alvarez, G. P. Siegal, and D. T. Curiel. 1998. Intracellular expressionof a single-chain antibody directed against human papillomavirus type 16E7 oncoprotein achieves targeted antineoplastic effects. Cancer Res58:1893-1900.) The specificity and sensitivity of anti-HERV-K-positiveclones against HERV-K env proteins were tested by an ELISA as describedpreviously. In brief, HERV-K env proteins (10 μg per mL, 100 μL perwell) or HERV-E env proteins (as controls) were coated in wells of96-well plates. The supernatants obtained from several positive clones(1:50 to 1:109,350 dilution with PBS) were added to the coated wells andincubated for 1 h at ambient temperature. HRP-conjugated antimouse IgGantibody (100 μL, 1:2000 dilution) was added to each well for another 1h at ambient temperature. The color was developed, and the plate wasread on a microplate reader at 405 nm. ELISA was also used to detectvarious anti-HERV antibodies in human sera, as described previously.Anti-human IgG antibody was used as a negative control, and anti-RGS mAb(Qiagen, Inc.; used to detect 6-His protein produced from pQE30 vector)or anti-HERV-K mAb was used as a positive control. The cut-off value fora negative reaction was 0.5 OD at 405 nm. The means of the thresholdvalues were used for the final analysis. For Western blot, purifiedHERV-K proteins (20 μg/well) were loaded onto 10-15% SDS gels. Aftertransfer to membranes, mAbs (1:1,000 dilution) or human sera (1:200dilution) were used as primary antibodies and incubated overnight at 4°C. Anti-mouse or human IgG HRP mAb (1:1,000 dilution) was added andincubated at room temperature for 1 hr and visualized using ECL(Upstate). Anti-RGS mAb (1:1,000 dilution, which detects 6-His proteinproduced from pQE30 vector) or anti-GST mAb (1:1,000 dilution, whichdetects GST protein produced from pGEX-4T1 vector) was used as apositive control.

Preparation of DC and EBV-Transformed Lines from Human PBMC. DC weregenerated from adherent or CD14-positive PBMC isolated by magnetic cellsorting with CD14 MicroBeads (Miltenyi Biotec, Auburn, Calif.). Theisolated cells were incubated for 6 days with GM-CSF and IL-4. After the6-day incubation period, the immature DC were harvested and transfectedwith K-SU protein or control proteins by DOTAP, and TNF-α was added tothe medium to obtain mature DC. To generate EBV-induced B lymphoblastoidlines (B-LCL) CD3⁺ cells were removed from PBMC by magnetic cell sortingwith an autoMACS separator (Miltenyi Biotech) using human CD3 beadsaccording to the manufacturer's instructions. B95-8 culture supernatantscontaining the transforming strain of EBV were then used to establishthe B-LCL.

FACS analysis of DC phenotypes. Immature or mature DC with or withoutprior pulsing with HERV-K env protein were phenotyped on day 7 using amulticolor CD86-PE/CD209-PerCP-Cy5.5/CD83-APC kit (BD Biosciences, SanJose, Calif.) and analyzed on a BD FACSCalibur system. DC stained withonly secondary antibody served as negative controls.

In vitro sensitization of T cells and T-cell proliferation assay. DCwere pulsed with antigen and matured as described above. Autologous PBMC(1×10⁶ cells/mL) were added to the loaded DC at a DC to PBMC ratio of1:30 on day 0. Recombinant human IL-2 (10 U/ml) was added and thecultures incubated for 7 days to generate 1-week IVS cells. The 1-weekIVS cells were restimulated on day 14 with autologous DC previouslypulsed with antigen to produce 3-week IVS cells. T cell proliferationwas evaluated in the PBMC or IVS cells that were stimulated with DC(pulsed for 72 hr with no added protein, K-SU protein or E6 protein, ata DC to PBMC or IVS ratio of 1:30. Results are expressed as counts perminute per 1×10⁵ PBMC or IVS cells.

ELISPOT assays. A GrB ELISPOT assay to detect and quantitatecytokine-secreting cells in response to antigen was performed using acommercial kit (Biosource International, Camarillo Calif.), followingthe manufacturer's instructions. The spots were evaluated using anautomated ELISPOT reader system (Carl Zeiss, Thornwood, N.Y.) with KSELISPOT software 4.5+ (ZellNet Consulting). Only spots with fuzzy ordiffuse borders were scored as positive. Net frequencies of spot-formingcells were calculated.

Cytotoxic T-lymphocyte assay. CTL assays were performed inround-bottomed 96-well plates using a standard 4-h ⁵¹Cr-release assay(Dolbier, C. L., R. R. Cocke, J. A. Leiferman, M. A. Steinhardt, S. J.Schapiro, P. N. Nehete, J. E. Perlman, and J. Sastry. 2001. Differencesin functional immune responses of high vs. low hardy healthyindividuals. J Behav Med 24:219-229) using 1×10⁵ target cells. Targetcells were either MCF-7 BC cells or HERV-K (or control antigen)transduced autologous DC or B-LCL cells. Unlabeled K562 cells (1×10⁵cells/well) were added to assess nonspecific lysis. To blockcytotoxicity, effector cells were pre-incubated for 30 min at ambienttemperature with an anti-human CD3 mAb (10 μg/mL; Ortho PharmaceuticalCorp, Raritan, N.J.).

Multiplex cytokine bead array analysis. The supernatants obtained from Tcell proliferation were collected after 7 days of IVS and stored at −20°C. for cytokine bead array analysis using a LINCOplex multipleximmunoassay-based protein array system (LINCO Research, St. Charles,Mo.), which contains microspheres conjugated with mAb specific fortarget proteins. Fluorescence intensity was measured using a Luminex 100instrument (Luminex Corporation, Austin, Tex.).

Intracellular cytokine staining. Cytokines produced by CD4⁺ and CD8⁺ Tcells, including TNF-α, IL-2, and IFN-γ, were assayed by cytokine flowcytometry as previously described (Martins, S. L., L. S. St John, R. E.Champlin, E. D. Wieder, J. McMannis, J. J. Molldrem, and K. V.Komanduri. 2004. Functional assessment and specific depletion ofalloreactive human T cells using flow cytometry. Blood 104:3429-3436;Komanduri, K. V., M. N. Viswanathan, E. D. Wieder, D. K. Schmidt, B. M.Bredt, M. A. Jacobson, and J. M. McCune. 1998. Restoration ofcytomegalovirus-specific CD4+ T-lymphocyte responses after ganciclovirand highly active antiretroviral therapy in individuals infected withHIV-1. Nat Med 4:953-956.). The activated cells were permeabilized withBD Cytofix/Cytoperm buffer for subsequent intracellular staining withPE-conjugated anti-TNF-α, anti-IL-2, or anti-IFN-γ (BD Pharmingen).APC-labeled anti-IgG2a or IgG1 and a PE-conjugated isotype controlcocktail were used as single color controls. The samples were acquiredand analyzed on a FACSCalibur system.

Statistical analysis. Each assay was performed in triplicate.Statistical significance between groups was determined by the unpaired,two-tailed student's t-test using Prism software (GraphPad). To compareHERV-K expression during progression from normal to cancerous, we usedChi-square test. A P value of <0.05 indicated a significant differenceamong treatments.

Example 21 Expression of HERV in Melanoma Cell Lines

Detection of the Env region of HERV transcripts in melanoma cells. TotalRNA was isolated from cells or tissues and treated with DNase to removeDNA contamination. Reverse transcription and PCR amplification wereperformed by standard protocols using various HERV Env outer sense andantisense primer pairs including ERV3, HERV-E4-1 and HERV-K type 1, asdescribed previously, and HERV-K type 2.

Expression of HER V-K env protein in melanoma biopsies. We havegenerated several anti-HERV-K env protein mAb (including 6H5, 4D1, 6E11,4E11 and 6E5) in our laboratory against recombinant HERV env proteinproducts such as HERV-K Env surface protein, Np9 and Rec proteins (seeFIG. 32). Anti-HERV-K Env protein mAb 6H5 was used to detect theexpression of HERV-K env protein in melanoma biopsies. More than 75% ofmelanoma biopsies were HERV-K positive (Table 8 and FIG. 33). Strongerexpression of HERV-K was detected as the Clark's level and Breslowthickness increased. All metastatic lymph nodes stained positive forHERV-K expression, even though some skin biopsies were negative. In allof the positive skin expression cases, at least 40% of tumor cells wereHERV-K positive.

In summary, the extent of expression of HERV-K is associated withseverity of melanoma. We observed no expression in melanoma in situ andnor-tumorigenic melanoma. There was focal expression in the deep tumorcluster of tumorigenic invasive melanoma in complex primary melanoma,where histology showed a tumorigenic compartment adjacent to anon-tumorigenic compartment. When the melanoma progressed to thevertical growth phase there were more positive cases with the strongestHERV-K staining in two desmoplastic types (spindle cell type).

TABLE 8 The expression profile of HERV-K env protein in melanomabiopsies HERV-K HERV-K % Metastasis LN skin HERV-K No. ARS¹ Primary toLN² histology type Breslow Clark expression expression positive 1 61WMSkin Superficial sprd³ 2 II 0-1⁴ 2 63WF Skin Superficial sprd 0.4 I 0 361M Skin nodular type 4 IV 2 to 3 40% 4 62WM Skin  N2⁵ nodular type 8 IVPositive 3 70% 5 29WM Skin nodular type >10 V 2 >70% 6 35WF Skin N1Superficial sprd <2 Positive 0 7 60HM Skin spindle cell V 3 70% 8 58HFN2 Positive 2 to 3 70% 9 50HM Skin N3 nodular type Positive 1 40% 10 61MEyelid nodular type 2 IV 1 11 70WM Skin nodular type 10 IV 3 >70% 1272HM Skin spindle cell >10 V 3 50% ¹ARS: Age, Race, and Sex. ²LN: lymphnodes. ³Superficial sprd: Superficial spreading. ⁴0: no expression ofHERV-K; 1: low expression; 2: intermediate expression; 3: strongexpression. ⁶N: number of lymph nodes affected: N1, 1-4 LN; N2, 4-9 LN;N3, >10 LN. Case No. 1 is negative with a few deeply invaded tumorclusters staining positive.

Detection of anti-HERV antibodies in melanoma patients. Patient sera wasserially diluted and tested by ELISA using recombinant purified fusionproteins. Approximately 90% of melanoma patients (37/41 patients) hadelevated anti-Rec titers, 85% (35/41) had elevated anti-Np9 titers, and39% (16/41) had elevated anti-HERV-K antibody titers (FIG. 34A). Incomparison with other human cancers such as colon cancer and breastcancer, melanoma patients had the highest antibody titers against bothRec and Np9 protein (FIG. 34B).

Induction of immune response in human cells. We have tested for thepresence of anti-HERV-K T-cell responses in human PBMC in cancerpatients and normal donors. We used an in vitro system to determine if aCTL immune response can be elicited in cancer patients. DCs weregenerated from adherent PBMC in cultures containing the cytokinecombination of GM-CSF and IL-4. Immature DCs were pulsed with or withoutHERV-K proteins (Kpro) or RNA (KRNA) and TNF-α for maturation. (FIG.35).

Examples Summary

Overall, we found that one human retroviral gene product, HERV-K env, isproduced as a full-length protein in many BC cell lines and primaryhuman BC specimens and is not found in normal tissue. HERV-K protein wasdetected in 85% of invasive ductal carcinomas stained by IHC. Inaddition, immunofluorescence microscopy and FACS analysis demonstratedthat the HERV-K env product is not only expressed in the cytoplasm of BCcells, but is also present as a transmembrane cell surface proteindetectable in non-permeabilized cells. The expression of HERV-K in BCwas found to be associated with the presence of HERV-K-specific CD8⁺T-cell responses in patient PBMC-derived T cells, while healthy donorsdid not exhibit considerable anti-HERV-K activity. These responses weremanifested in T cell proliferation, GrB secretion, elevated Th1 cytokine(IL-2 and IFN-γ) secretion, and lysis of HERV-K⁺ target cells,suggesting that T cells in these patients had been primed in vivo toHERV-K env protein derived from their tumors.

HERV-K env protein may overcome the immunosuppression that is observedin cancer patients. Helper T cell activation results in secretion ofinterleukin-2 (IL-2), which augments CTL response, and we show increasedIL-2 secretion in BC patient K-SU stimulated IVS cells. Our results alsoshow significantly decreased IFN-γ secretion in BC patients relative tonormal female donors, which was reversed by K-SU stimulation of BCpatient IVS to give increased IFN-γ secretion relative to normal controlfemales, who showed no response to K-SU stimulation.

The cellular immune responses were induced by HERV-K env protein, andnot by other viral proteins produced by the same expression vector, suchHPV16 E6, HPV16 E7 or LMP2A. The HERV-K specific T cell immuneresponses, including CD4⁺ and CD8⁺ T cell responses, were likely due toHERV-K env protein itself and not to bacterial contamination for tworeasons. First, HERV-K mRNA produced by in vitro transcription,independent of expression in a bacterial system, also induced immuneresponse relative to control proteins. Second, control HPV16 E6 andHPV16 E7 proteins produced in bacteria did not promote immune responseto nearly as great an extent as did HERV-K protein. Importantly, theimmune response could be induced by only a single in vitrosensitization, which led to a recall response.

Only 23.5% of BC patients (4/17) secreted IFN-γ at levels ≧50 pg/mLbefore HERV-K in vitro sensitization. After IVS, more than 94% of the BCpatients (16/17) had increased IFN-γ secretion. The production of IFN-γin response to HERV-K is significant because IFN-γ secretion at levels≧50 pg/mL in response to TAA in PBMC from cancer patients was previouslyreported to be associated with an increase in median cancer patientsurvival of 88 to 470 days. The production of IL-2, which is used incancer vaccines to boost immune response to specific cancer antigens,was also significantly increased in BC patients after IVS (71.89±23.06pg/ml) compared to levels before IVS (12.89±2.242 pg/ml; N=17) (FIG.6C). These results suggest that HERV-K env surface protein activates animmunostimulatory response against neoplasms.

CD8⁺ T cells have traditionally been the main focus of tumorimmunologists developing anti-cancer vaccines. However, more recentlythe critical role of antigen-specific CD4⁺ T-cell responses ingenerating more effective anti-tumor responses has been recognized. Thisappreciation for the role of CD4⁺ T cells stemmed from the discovery ofantibodies in patients against tumor antigens including cancer testisantigens such as MAGE-3 and NY-ESO-1 using “SEREX,” and theidentification of HLA class II-binding epitopes from the same tumorantigens that are recognized by CD4⁺ T cells. Optimal anti-tumorresponses against a specific TAA have been found in cases where bothCD8⁺ and Th1 CD4⁺ tumor antigen-specific responses are generated. Theexpression of HERV-K at the surface of BC cells also suggests that theprotein can be shed from and internalized by B cells and trigger CD4⁺T-cell responses and HERV-K-specific IgG production. Indeed, we foundsignificant titers of anti-HERV-K env IgG in the sera of BC patientswhile insignificant titers were found in normal donor sera. The presenceof these antibodies also suggests that soluble retroviral envelopeproteins such as HERV-K may circulate in the blood of cancer patientsand may be a diagnostic marker for BC. The presence anti-HERV-K IgG isindicative of the activation of CD4⁺ T-helper cells. The activation ofCD4⁺ T-helper cells along with CD8⁺ T cells against HERV-K issignificant especially in light of the growing importance of T-helpercells in driving and maintaining CTL responses through the provision ofcytokines and signals activating DC antigen presentation to CD8⁺ Tcells.

The precise mechanism by which cancer cells respond to processedpeptides of HERV-K env proteins has not been elucidated. The presence ofgene polymorphisms or sequence variants, which are exemplified by theexpression of HERV-K-MEL in melanoma tumors and HERV-K in BC, and thepresence of variants of human teratocarcinoma-derived virus(HTDV)/HERV-K in teratocarcinoma cell lines, may help explain theresponse of cancer cells to HERV-K peptides. Importantly, multipleHERV-K env spliced products obtained from human BC tissues encode openreading frames without stop codons, suggesting the opportunity fortranslation of variant HERV-K proteins and subsequent processing oftheir peptides. Thus, T cell responses against multiple HERV-derivedepitopes may exist in cancer patients. Anti-HERV-K antibody can inhibitBC cell, but not benign breast cell proliferation and induce BC cells toundergo apoptosis (data not shown). These data support the idea thatHERV-K env surface protein and its antibody suppress cancer throughseveral mechanisms.

Moreover, the examples provide direct evidence that HERV-K Env proteinsare immunogenic in melanoma patients. Importantly, these studies are thefirst to directly show that these viral antigens induce T-cell and CTLcapable of killing HERV-K-expressing target cells. We have also foundthat PBMC from ovarian and breast cancer patients secrete several Th1 orTh2 cytokines in response to HERV-K antigens.

In summary, we report here for the first time the presence of cellularand humoral immune responses against a human endogenous retroviral envprotein in breast cancer, ovarian cancer, and melanoma and its abilityto recognize and kill cancer cells in vitro. HERV-K env proteinexpression was found to be highly expressed specifically in cancer withprotein expression in >80% of human breast ductal carcinomas and noexpression in normal tissues. The re-activation of endogenous retroviralgene products and synthesis of mature protein products in cancer makesthese a potentially valuable new pool of tumor associated antigens fortargeting in therapeutic vaccines in cancers. The silencing ofretroviral gene expression throughout our lifetime and theirreactivation specifically in cancer cells suggests that immunologicalself-tolerance mechanisms against HERV-K and other retroviral proteinsmay be limited or that the immune system exists in a state of ignorancetowards these antigens until cancer develops. Accordingly, prophylacticvaccination may be used to prevent primary tumor development. Similar toanti-viral vaccines now used to prevent cervical cancer and other tumorssuch as liver cancer, prophylactic vaccines against non-expressedretroviral antigens may elicit long-lived retroviral antigen-specific Tcell responses (T-central memory) in an otherwise ignorant host that caneradicate early malignancies re-expressing these retroviral geneproducts.

Additional Data

Monoclonal, chimeric, humanized, primatized, single chain, Fab fragmentsand other similar types of antibodies are not found naturally in humans.While HERV-K positive cancer patients may have anti-HERV-K antibodies,until this teaching, no antibody has been discovered, isolated and/ortested for their antitumor effects, especially the antibody that bindsHERV-K env proteins expressed only on cancer cells and tumor biopsiesand as obtained from HERV-K⁺ cancer patients.

For example, monoclonal and scFv antibodies are only produced in miceusing HERV-K env recombinant protein obtained from tumor biopsies madein the lab by molecular biology techniques. Also, multiple copies HERV-Kgenes exist in the human genome, but not every gene copy can be activelytranscribed or translated into proteins. Several hundred HERV-K envcDNAs isolated from human tumor biopsies have been sequenced andselected to generate our antibodies. The monoclonal antibodies wereselected and tested for their specificity and sensitivity against HERV-Kenv protein by multiple assays and five clones were chosen to test forantitumor effects in vitro and in vivo. In addition, scfv for theantibody identified herein as 4D1 with sequences provided in FIG. 16 andthe antibody identified herein as 6H5 with sequences provided in FIG. 17were both generated by a multiple panning process and functionallyselected. Their CDRs for heavy chain and light chain are as shown inFIGS. 36 and 37 respectively. More specifically, FIG. 36 depicts the 4D1scFv nucleotide and amino acid sequences. The 4D1 scFv nucleotidesequence illustrated in FIG. 36 is comprised in SEQ ID NO 11 whichsequence includes the consensus sequence sites for two restrictionenzymes, Sfi I and the NotI, at the 5′ and 3′ ends respectively. SEQ IDNO: 13 also comprises a 4D1 scFv nucleotide sequence as shown in FIG. 36but is missing the 5′ Sfi I consensus sequence. The amino acid sequencesdepicted in FIG. 36 correspond to the amino acids of SEQ ID NO: 12 (withboth the restriction sites, 251 amino acid long) and SEQ ID NO:14 (withonly the Not I restriction site, 247 amino acids in length). Alsodepicted in FIG. 36 (from nucleotide 1-337) is the nucleotide sequenceof SEQ ID NO: 15, which comprises the heavy chain variable region of 4D1scFv. The amino acid sequence of SEQ ID NO: 16 (112 amino acids long)corresponds to the protein encoded by SEQ ID NO: 15.

FIG. 36 further shows the nucleotide sequence of SEQ ID NO: 17 (303nucleotides long), which is the light chain variable region of 4D1 scFv.The amino acid sequence of SEQ ID NO: 18 (101 amino acids long)corresponds to the protein encoded by SEQ ID NO: 17. The three heavychain complementarity determining regions (CDR) of 4D1 scFv are depictedin FIG. 36, highlighted in blue and denoted as CDR-H1 (SEQ ID NO: 19(nucleotide); SEQ ID NO: 20 (amino acid); CDR-H2 (SEQ ID NO: 21(nucleotide); SEQ ID NO: 22 (amino acid); and CDR-H3 (SEQ ID NO: 23(nucleotide); SEQ ID NO: 24 (amino acid). The three light chain CDRs of4D1 scFv are also depicted in FIG. 36, highlighted in yellow and denotedas CDR-L1 (SEQ ID NO: 25 (nucleotide); SEQ ID NO: 26 (amino acid);CDR-L2 (SEQ ID NO: 27 (nucleotide); SEQ ID NO: 28 (amino acid); andCDR-L3 (SEQ ID NO: 29 (nucleotide); SEQ ID NO: 30 (amino acid).

FIG. 37 depicts the 6H5 scFv nucleotide and amino acid sequences. The6H5 scFv nucleotide sequence of FIG. 37 is comprised in SEQ ID NO 49.The entire amino acid sequence depicted in FIG. 37 corresponds to theamino acids of SEQ ID NO: 50. Also depicted in FIG. 37 is the nucleotidesequence of SEQ ID NO: 51 (334 nucleotides in length), which is theheavy chain variable region of 6H5 scFv. The amino acid sequence of SEQID NO: 52 (111 amino acids long) corresponds to the protein encoded bySEQ ID NO: 51.

FIG. 37 also shows the nucleotide sequence of SEQ ID NO: 53 (310nucleotides long), which is the light chain variable region of 6H5 scFv.The amino acid sequence of SEQ ID NO: 54 (103 amino acids long)corresponds to the protein encoded by SEQ ID NO: 53. The three heavychain complementarity determining regions (CDR) of 6H5 scFv are depictedin FIG. 37, highlighted in blue and denoted as CDR-H1 (SEQ ID NO: 55(nucleotide); SEQ ID NO: 56 (amino acid); CDR-H2 (SEQ ID NO: 57(nucleotide); SEQ ID NO: 58 (amino acid); and CDR-H3 (SEQ ID NO: 59(nucleotide); SEQ ID NO: 60 (amino acid). The three light chain CDR of6H5 scFv are also depicted in FIG. 37, highlighted in yellow and denotedas CDR-L1 (SEQ ID NO: 61 (nucleotide); SEQ ID NO: 62 (amino acid);CDR-L2 (SEQ ID NO: 63 (nucleotide); SEQ ID NO: 64 (amino acid); andCDR-L3 (SEQ ID NO: 65 (nucleotide); SEQ ID NO: 66 (amino acid). FIG. 68depicts the humanized 4D1 scFv (Hu 4HD1 scFv) nucleotide and amino acidsequences. The Hu 4D1 scFv nucleotide sequence of FIG. 68 is comprisedin SEQ ID NO 31.

The entire amino acid sequence depicted in FIG. 68 corresponds to theamino acids of SEQ ID NO: 32 (254 amino acid long). Also FIG. 68 showsthe nucleotide sequence corresponding to SEQ ID NO: 33, which is theheavy chain variable region of Hu 4D1 scFv. The amino acid sequence ofSEQ ID NO: 34 comprise the protein encoded by SEQ ID NO: 33. FIG. 68further identifies the nucleotide sequence of SEQ ID NO: 35 (327nucleotides long), which is the light chain variable region of Hu 4D1scFv. The amino acid sequence of SEQ ID NO: 36 (109 amino acids long)corresponds to the protein encoded by SEQ ID NO: 35. The three heavychain complementarity determining regions (CDR) of Hu 4D1 scFv aredepicted in FIG. 68, highlighted in blue and denoted as muCDR-H1 (SEQ IDNO: 37 (nucleotide); SEQ ID NO: 38 (amino acid); muCDR-H2 (SEQ ID NO: 39(nucleotide); SEQ ID NO: 40 (amino acid); and muCDR-H3 (SEQ ID NO: 41(nucleotide); SEQ ID NO: 42 (amino acid). The three light chain CDR ofHu 4D1 scFv are depicted in FIG. 68, highlighted in yellow and denotedas muCDR-L1 (SEQ ID NO: 43 (nucleotide); SEQ ID NO: 44 (amino acid);muCDR-L2 (SEQ ID NO: 45 (nucleotide); SEQ ID NO: 46 (amino acid); andmuCDR-L3 (SEQ ID NO: 47 (nucleotide); SEQ ID NO: 48 (amino acid).

FIG. 69 depicts the humanized 6H5 scFv (Hu 6H5 scFv) nucleotide andamino acid sequences. The Hu 6H5 scFv nucleotide sequence of FIG. 69 iscomprised in SEQ ID NO 67. The entire amino acid sequence depicted inFIG. 69 corresponds to the amino acids of SEQ ID NO: 68 (254 amino acidlong). Also FIG. 69 identifies the nucleotide sequence corresponding toSEQ ID NO: 69 (328 nucleotides), which is the heavy chain variableregion of Hu 6H5 scFv. The amino acid sequence of SEQ ID NO: 70 (109amino acids) comprise the protein encoded by SEQ ID NO: 69. FIG. 69shows the nucleotide sequence of SEQ ID NO: 71 (334 nucleotides long),which have the light chain variable region of Hu 6H5 scFv. The aminoacid sequence of SEQ ID NO: 72 (111 amino acids long) corresponds to theprotein encoded by SEQ ID NO: 71. The three heavy chain complementaritydetermining regions (CDR) of Hu 6H5 scFv are also depicted in FIG. 69,highlighted in blue and denoted as muCDR-H1 (SEQ ID NO:73 (nucleotide);SEQ ID NO: 74 (amino acid); muCDR-H2 (SEQ ID NO: 75 (nucleotide); SEQ IDNO: 76 (amino acid); and muCDR-H3 (SEQ ID NO: 77 (nucleotide); SEQ IDNO: 78 (amino acid). The three light chain CDR of Hu 6H5 scFv arefurther depicted in FIG. 69, highlighted in yellow and denoted asmuCDR-L1 (SEQ ID NO: 79 (nucleotide); SEQ ID NO: 80 (amino acid);muCDR-L2 (SEQ ID NO: 81 (nucleotide); SEQ ID NO: 82 (amino acid); andmuCDR-L3 (SEQ ID NO: 83 (nucleotide); SEQ ID NO: 84 (amino acid).

Western blot using 6H5 mAb revealed that HERV-K env protein wasexpressed only in breast cancer (SKBR3, T47D, MDA-MB-231 and MCF-7).Expression in breast cancer was greater than in early transformed breastcell lines (MCF-10AT), and was not observed at all in an immortalizednon-neoplastic breast epithelial cell line (MCF-10A) (FIG. 38). HERV-Kenv protein in breast cancer cells was sensitive to PNGase F cleavage(FIG. 39; Pink gel), leading to a shift in the mobility of the bandrecognized by 6H5 (Blue gel), and confirming that HERV-K env protein isa glycoprotein. The antibodies detected the expression of HERV-K envprotein only in cancer tissues (including breast, colon, lung, lymphoma,melanoma, and prostate, with the exception of brain tumor), but not innormal (no expression in a total of 38 normal tissues obtained from 33organs; FIGS. 41 to 43) or uninvolved normal tissues adjacent to cancertissue array samples stained with 6H5 under identical stainingconditions (FIG. 40 and Table 9). This array contained 492 tissues on asingle slide. Until this teaching, no one has yet reported that HERV-Kenv protein is expressed in other cancers, including lung (Table 10 and11), colon (FIG. 44 top), pancreas (FIG. 44 bottom), and others in FIG.44 and Tables 9, 10 and 11). Table 12 shows the expression profile ofHERV-K in melanoma biopsies.

The antibodies disclosed herein prevent the proliferation of an alreadyexisting cancer cell, and, therefore, prevent the proliferation ofcancer cells from forming cancer tissues. Furthermore, these antibodiesbind to the HERV-K antigen on the cell surface of any HERV-K⁺ cancerincluding breast (FIG. 45), melanoma (FIGS. 46 and 47), colon, ovarian,lung, liver, pancreatic, and others (FIG. 48). HERV-K negative cellswere detected in most non-malignant cells including breast cell linesMCF-10A, MCF-10AT, and ovarian cell lines T80, T29, and T72 (data notshown). The numbers of surface molecules of HERV-K env protein in breastcancer cells and non-cancer breast cells was quantified by flowcytometry with a QIFI kit using calibration beads (FIGS. 45, 47 and 48),dry cell ELISA (FIG. 46 left), or immunofluorescent staining (FIG. 46right). Cell surface expression of the HERV-K antigen in several typesof cancer cells has also been shown. Specifically, low surfaceexpression of HERV-K env protein was found in LS174T and CaCO2 coloncancer cells.

The ability of HERV-K recombinant protein to block binding ofanti-HERV-K antibodies to the cell surface was evaluated bypre-incubating the mAbs with HERV-K recombinant protein (FIG. 49).Anti-mIgG AF647 only was used as control. Furthermore, cycling of HERV-Kenv protein was observed between the cell surface and intracellularstores in breast cells. Cells were first incubated with 6H5 mAb at 4°C., and HERV-K env protein membrane expression at 0 time was determined.After 1, 5, 15, and 45 min incubation at 37° C., samples were evaluatedfor membrane expression to assess time-dependent endocytosis of HERV-Kenv protein. Half of the cells were labeled with anti-mouse-IgG AF647 todetect HERV-K env protein remaining on the cell surface (top panel). Theother half were treated with an acid buffer to strip the 6H5 from themembrane, and were re-incubated with another anti-HERV-K env mAb,4D1-555, to detect the change in HERV-K cell-surface binding level(bottom panel). Time-dependent internalization of HERV-K env protein wasobserved on MCF-7, MDA-MB-231, and T47D breast cancer cells (FIG. 50).The disappearance of 6H5 was presented as percentage of internalizationcalculated by the following ratio: (mean fluorescence at 0 min-meanfluorescence at each time point)/(mean fluorescence at 0 min). Thepercentage of internalization at 15 and 45 min was 36.6% and 39% forT47D cells, 47.96% and 57.92% for MCF-7, and 41.94% and 64.52% forMDA-MB-231 cells, respectively. No significant change in cell-surfacebinding level of the conformation-dependent 4D1 antibody was detected(bottom panel). Furthermore, net cellular uptake rates of anti-HERV-Kantibodies were determined in HERV-K⁺ cells (FIG. 51). Surface quenchingallows for distinction of surface and internal antibody fractions. Totalcellular fluorescence was measured at each time point by flow cytometryand the internal and surface fractions determined by surface quenchingwith an anti-Oregon green IgG. The internal fluorescence was thencalculated as total MFI-surface MFI. Total, surface, and internalizedHERV-K env protein in MCF-7, MDA-MB-231, T47D, and MCF-10A cells werecompared at 0, 1.5, 3, 6, and 9 hr. The rank in expression of HERV-K envprotein from high to low was MCF-7, MDA-MB-231, T47D, and MCF-10A. Inaddition, metabolic turnover of HERV-K env protein was assayed todetermine the HERV-K env protein internalization rate (FIG. 52). HERV-Kwas degraded with a half amount between 15 to 45 min, similar to theinternalization rate of the anti-HERV-K antibodies. Of interest, anincrease in surface expression of HERV-K env protein was detected at 180min. The two bands of surface proteins detected correspond to type 2(top band) and type 1 of HERV-K env protein. The difference in band sizeis due to the presence or absence of a 292 bp sequence in the env gene.

MCF-7 cells were treated with several concentrations of 6H5 or 6E11 mAbor mIgG on day 0, and cell proliferation was measured by MTS assay (OD492 nm; top left) or cytotoxicity assay (crystal violet staining; OD 600nm; top right) after 72 hr (FIGS. 53 and 54). 6H5 and 6E11 inhibitedMCF-7 cell proliferation, compared to cells treated with mIgG. Inaddition, both mAbs were cytotoxic toward the breast cancer cell lines,compared to cells treated with mIgG. HERV-K 6H5 and 6E11 mAbs (≦0.1μg/ml) inhibited MCF-7 cell proliferation (p<0.0001). Both 6H5 and 6E11also showed significant cytotoxicity toward MCF-7 cells (p=0.0069 for6H5; p=0.0002 for 6E11). There was no cytotoxicity of 6H5 toward MCF-10Anormal breast cells, in contrast to the significant cytotoxicity of 6H5toward MCF-7 and MDA-MB-231 breast cancer cells (FIG. 54).

As to cell death, FIGS. 53, 54, 55 and 56 provide data showingconclusively that the antibodies of the subject invention inducesapoptosis (cell death). The annexin V assay allows for the rapid,specific, and quantitative identification of apoptosis in individualcells. Viable cells are Annexin V− and 7AAD−; cells in early apoptosisare Annexin V+ and 7AAD−; dead cells or cells in late apoptosis areAnnexin V+ and 7AAD+. Untreated cells or cells treated with mIgG wereused as controls. The effect of 6H5 on induction of apoptosis inmelanoma cells is shown in FIG. 55. The effect of 6H5 (red tracing) or6H5-rGel (blue tracing) on induction of apoptosis in breast cells, incomparison to the same cells not treated with 6H5 or 6H5-rGel (cellsstained with anti-mouse IgG; gray color) is shown FIG. 56. The breastcancer cell lines MDA-MB-453 (52.28% or 33.82% apoptosis, aftertreatment with 6H5 or 6H5-rGel, respectively), MCF-7 (43.68% or 39.32%),T47D (33.14% or 23.29%), and MDA-MB-231 (63.6% or 53.02%) were inducedto undergo apoptosis to a much greater extent than MCF-10A non-malignantbreast (6.48% or 4.96%) or MCF-10AT preneoplastic breast (8.4% or 9.42%)cells, after 6H5 (10 □g/ml) or 6H5-rGel (10 □g/ml) treatment. This6H5-induced apoptosis is followed by the elimination of dead cells andthereby eliminates cancer cells.

These anti-HERV mAbs affect expression of a major death receptor onbreast cancer cells. Caspases play an important role in programmed celldeath. Caspase-3 is a key executioner of apoptosis, whose activation ismediated by the initiator caspases, caspase-8 and caspase-9. There wasincreased expression of active caspases 3, 8, and 9 in breast cancercells treated with 6H5 or 6E11 compared with cells treated with controlmIgG (FIG. 57). Expression of these caspases in breast cancer cellstreated with 6E11 or 6H5 did not decrease after treatment with Z-VAD(150 μM), which indicates that 6H5 and 6E11 induce breast cancer cellsto undergo apoptosis through caspase 3 independent pathways.

BrdU is incorporated instead of thymidine into the DNA of proliferatingcells and subsequently detected by fluorescent activated cell sorting(FACS) using a Beckton Dickinson FACSarray instrument. Cells weretreated with 6H5 (10 μg/ml) for 72 h and DNA-BrdU incorporation wasdetermined (FIG. 58). BrdU incorporation in breast cells was expressedas a percentage of control (the cells treated with mIgG). BrdUincorporation in all cancer cells was lower than MCF-10A (non-malignantbreast cells). Breast cancer cells treated with 6H5 revealed atime-dependent decrease in DNA synthesis in various breast cancer celllines (data not shown). HERV-K env protein activation and cell cycleresponses were also observed after treatment with the mAbs. To testwhether an induction of cell cycle arrest contributed to theantiproliferative potency of 6H5 in breast cancer cell lines, weperformed cell cycle analyses. Incubation breast cancer cells with 10μg/ml for 24, 48, 72 hr led to arrest of the cells in various phases.For example, G0/G1 arrest was found in T47D breast cancer cells treatedwith 6H5, whereas S arrest was observed for MCF-7 breast cancer cells,and G2 arrest for other breast cancer cells (FIG. 59).

IgG type antibodies containing the Fc portion usually mediateimmunological cytotoxicity via antibody-dependent cell-deathcytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). MurineIgG2a and IgG3 mAbs are the most effective in mediating ADCC, and both6H5 and 6E11 are IgG2a. We demonstrated that both 6H5 and 6E11 arereactive against breast cancer cell lines in CDC (FIG. 60) and ADCC(FIG. 61) assays. There was a greater reduction in cell viability whenMCF-7 cells were treated with 10 μg 6E11 than with 1 μg, or when MCF-7cells transfected with HERV-K surface recombinant protein were treatedwith 10 μg 6E11 (10 μg+K) than with 1 μg (10 μg+K). In addition, cellviability decreased when the dilutions of complement were increased to1:10, 1:15 or 1:20, compared to controls (no mAb, or heat-inactivatedcomplement) (FIG. 60 top panel). A significant reduction in cellviability was observed in many instances when MCF-7 or MDA-MB-435eB1cells were treated with mAb in the presence of 1:10 to 1:30 dilutions ofcomplement, in CDC assays (FIG. 60; bottom left and right panels). Thepercent lysis in ADCC assays ranged from 15% to 30% after treatment with6H5 or 6E11, using normal donor PBMC (FIG. 61, top left and top rightpanels). No mAb (T+E) or no effector cells (T+Ab) were used as controls.Higher cytotoxicity toward MCF-7 was observed if we used PBMC frombreast cancer patients (BC4703; IDC) than from normal donors (NL5187 orNL3520) (FIG. 61, bottom panel). However, not every breast cancerpatient's PBMC had higher cytotoxicity toward MCF-7 (BC5796). Inaddition, we noted that increased cell lysis was detected in targetcells (T) that expressed HERV-K env protein by transfection. The aboveresults clearly demonstrate a biological effect of our HERV-K anti-tumorantibody. Our results and others suggest that HERV-K plays a role inregulation of cell growth and tumor progression, which prompted us toinvestigate the specific contribution of HERV-K env protein totumorigenicity in vivo. Tumor sizes were significantly reduced, and theappearance of tumors was significantly delayed, in groups ofimmunodeficient mice inoculated with MDA-MB-231 (FIG. 62, top left andtop right panels) and MCF-7 (top right panel), and treated with 6H5 only(top left and top right panels) or 6H5 and 6H5rGel (bottom panel). Asignificant tumor size reduction was observed in mice treated with 6H5(P=0.0051 for MCF-7 or P=0.0275 for MDA-MB-231; paired t-test), or with6H5 and 6H5-rGel (P=0.0020 for one-way analysis of variance or P<0.0001for Bartlett's test for equal variances). Tumor tissues obtained frommice treated with 6H5 for 0, 1 or 2 weeks were compared using TUNEL orKi-67 assays. As shown in FIG. 63, there were 2.5 and 3.6-fold increasesin TUNEL-positive cells, and a 29.3 and 60.3% decrease in Ki-67-positivecells, in tumors from mice treated with 6H5 after 1 week and 2 weekperiods, respectively, in comparison to control mice (FIG. 7 d;P<0.0001, one way analysis of variance). In general, there was decreasedexpression of HERV-K env protein and Ki67, and increased apoptosis inbreast cancer cells treated with 6H5, and these effects were greaterwhen cells were treated twice with mAb.

Further, the epitopes of the antibodies of prior art such as Herve etal. are not the same as the epitopes described herein. Herve et al.found the antibodies in 32-47% of 84 sera from patients with autoimmunerheumatic disease, and 29% of 35 normal controls. Herve et al.discovered an immunodominant epitope (GKTCPKEIPKGSKNT) using patientserum, but Herve et al. did not isolate the antibody. While theantibodies of Herve et al. can be isolated, they cannot be modified toproduce the antibodies described herein. It is only through the teachingprovided in this specification that it is known which antibodies areeffective in treating cancer. Herve et al only reported on the presenceand epitopes of antibodies and such antibodies were not useful to treatdisease. This is especially true because Herve et al. did not isolatethese antibodies, which are contained in the milieu of many thousands ofproteins present in human cells. Herve et al. found that the antibodyexists in the sera of humans, which is not equivalent to isolating it.Multiple HERV-K alternatively-spliced proteins are present in humans,and we are the only group who has reported and isolated those HERV-Kvariants that are active in cancer.

In addition, Herve et al. does not present evidence that the antibodiesdisclosed could be used to treat any disease when administered toanother individual. Nor is there a reason to believe this. An antibodymay bind to an antigen on a cancer cell. However, it does not necessaryfollow that such antibody will kill the cancer cell. In fact, it isconceivable that binding will promote the growth of the cell viaactivation of growth-stimulatory cell signaling pathways. On the otherhand, as noted above, the antibodies described herein have been shownnot only bind to the antigen, but to promote cell death. For example,approximately twenty copies of endogenous betaretroviruses (enJSRVs) arepresent in the genome of sheep and goats, but only one sheep pulmonaryadenomatosis virus (Jaagsiekte sheep retrovirus) induces a naturallyoccurring lung cancer. More than 170 copies of HERV-K are present in thegenome of humans, but the expressed copies that induce naturallyoccurring cancer are not known yet. Five individual anti-HERV-Kantibodies were selected from a library and they have common propertiessuch as greater reactivity toward cancer cells than non-malignant cells;however, they have different sensitivity to various cell lines orepitopes of HERV-K env proteins. The role(s) of HERV-K play intumorgenesis are not clear. They are silent in normal, but expressed incancers to activate other genes.

For example, in our experiments, MCF-7 cells were treated with mIgG or6H5 (10 μg/ml) for 24 hr, and three Superarray PCR array kits were usedto determine how blocking of HERV-k with 6H5 affects the apoptosispathway, the cancer pathway, and the p53 signaling pathway (FIGS. 64, 66and 67). We found that 3 of 84 key genes involved in apoptosis, orprogrammed cell death were upregulated by 6H5 using a human apoptosisPCR array and the fold changes are shown (FIG. 64). The threeupregulated genes are TNFRSF25: tumor necrosis factor receptorsuperfamily, member 25; TNFSF8: tumor necrosis factor (ligand)superfamily, member 8; and CIDEA: cell death-inducing DFFA-like effectora. CIDEA was further confirmed by Western blot using anti-CIDEA antibody(FIG. 65). The expression of CIDEA was detected only in cancer cellstreated with 6E11, but not mIgG. In FIG. 66, TWIST1 (Probabletranscription factor) and MMP1 (collagenase-1), genes involved ininvasion and metastasis, were downregulated in MCF-7 breast cancercells, as assessed by the Cancer Finder Pathway Superarray, and the foldchanges are shown.

As shown in FIG. 67, changes in expression of 84 genes related top53-mediated signal transduction, including p53-related genes involvedin the processes of apoptosis, the cell cycle, cell growth,proliferation and differentiation, and DNA repair were evaluated usingthe p53 Signaling Pathway RT Profiler PCR Array. The array includesp53-related genes involved in the processes of apoptosis, the cellcycle, cell growth, proliferation and differentiation and DNA repair. Wefound that 10 of 84 genes were upregulated in the cells treated with6H5, in comparison to control cells treated with mIgG. The roles ofthese genes in relation to cancer are shown below:

WT1 2.31: Negative Regulation of the Cell Cycle: TP53 2.35: Induction ofApoptosis

TNFRSF10D 2.68: Anti-Apoptosis, TNF-related apoptosis-inducing ligand(TRAIL)MYOD1 2.95: Cell Growth and Differentiation, myogenic differentiation 1TNF 2.37: Anti-Apoptosis, TNF initiates programmed cell death (PCD) orapoptosis in transformed cells causing DNA fragmentation and cytolysisP53AIP1 3.59: Other Apoptosis Genes, p53-regulated Apoptosis-InducingProtein 1, p53AIP1, which is localized within mitochondria, leads toapoptotic cell death through dissipation of mitochondrial A(psi)m.p53AIP1 Regulates the Mitochondrial Apoptotic Pathway, p53AIP1 gene, anovel p53 target that mediates p53-dependent apoptosis

FASLG 3.86: Induction of Apoptosis

GML 3.67: Other Apoptosis Genes: Glycosyl-phosphatidylinositol-anchoredmolecule-like protein (GML) may play a role in the apoptotic pathway orcell-cycle regulation induced by p53 after DNA damageIFNB1 3.36: Negative Regulation of Cell Proliferation, interferon, beta1, fibroblast, has been shown to be frequently deleted or rearranged ina number of human cancers.IL6 3.05: Positive Regulation of Cell Proliferation: IL-6 was found toprotect against p53-induced apoptosis.

TABLE 9 Expression of HERV-K in a tissue array by IHC using 6H5 mAbTissues Negative Positive N Brain tumor 21 (100%) 0 21 Breast AdCa 3(7.14%) 39 (92.86%) 42 Colonic AdCa 0 12 (100%) 12 Lung cancer 4 (9.76%)37 (90.24%) 41 Lymphoma 33 (82.5%) 7 (17.5%) 40 Melanoma 4 (23.53%) 13(76.47%) 17 Prostate AdCa 0 3 (100%) 3 Normal tissues 38 (100%) 38 Totalcases 214

TABLE 10 Expression of HERV-K in a lung tissue array by IHC using 6H5mAb 3+ 2+ 1+ Neg Age f/m Total Adca 4*  3 6 2 60.12 6f/9m 15 squamous3   4 2 2 59.09  1f/10m 11 others 1   1 0 3 60.2 3f/2m 5 Adca 26.6720.00 40.00 13.33 86.67 squamous 27.27 36.36 18.18 18.18 81.82 others20   20 0 60 40.00 The expression of HERV-K env protein was evaluated in31 tumor cases and 31 matched normal tissues by immunohistochemistryusing 6H5 mAb. Results for matched normal tissues are not shown becauseonly 1 matching normal case (matched to Adca) was weakly positive Neg:Negative *Values are number of tissues in each category. Positivestaining was scored as 3 > 2 > 1 Adca: adenocarcinoma Squamous: squamouscell carcinoma Others: including carcinoid tumor, large cell carcinoma,large cell necroendocrine carcinoma, and bronchioloalveolar carcinoma

TABLE 11 Expression of HERV-K in a lung tissue array by IHC using 6H5mAb 3+ 2+ 1+ Neg Age f/m Total Adca 13*   7 4 3 58.59 16m/11f 27squamous 3   3 4 1 62.67 2f/9m 11 others 1   2 3 N/A 58.83 3f/3m 6non-neopla 7 59 2f/5m 7 Adca 48.15 25.93 14.81 11.11 88.89 squamous27.27 27.27 36.36 9.09 90.91 others 16.67 33.33 50.00 0.00 100.00 Theexpression of HERV-K env protein was evaluated in 44 tumor cases and 7normal lung tissues by immunohistochemistry using 6H5 mAb Neg: Negative*Values are number of tissues in each category. Positive staining wasscored as 3 > 2 > 1 Non-neopla: non-neoplastic Adca: adenocarcinomaSquamous: squamous cell carcinoma Others: including carcinoid tumor,large cell carcinoma, large cell necroendocrine carcinom, andbronchioloalveolar carcinoma

TABLE 12 The expression profile of HERV-K env protein in melanoma Insitu Non-nodular Tissue Nevi melanoma MM* Nodular MM Metastases #positive/# total 3/12 8/12 17/19 14/14 4/4 score ± SEM 0.42 ± 0.23 0.67± 0.49 1.95 ± 0.91 2.29 ± 0.61 1.25 ± 0.50 *MM: malignant melanoma

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

Therefore, the present disclosure is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Whilenumerous changes may be made by those skilled in the art, such changesare encompassed within the spirit of this disclosure as illustrated, inpart, by the appended claims.

1. An anti-HERV-K positive antibody.
 2. The antibody of claim 1 whereinthe antibody binds to HERV-K env protein.
 3. The antibody of claim 1wherein the antibody is a chimeric antibody.
 4. The antibody of claim 1wherein the antibody is a single chain antibody (scFv).
 5. An isolatedantibody that prevents HERV-K positive cancer cell proliferation.
 6. Anisolated antibody that induces HERV-K positive cancer cells to undergoapoptosis.
 7. A humanized antibody that binds to the HERV-K env proteinsexpressed from HERV-K positive cancer cells.
 8. An isolated antibodywhich binds to HERV-K env protein consisting of an amino acid sequenceat least 80 percent homologous to SEQ ID. NOS. 12, 14, 32, 50 or 68
 9. Amethod for preventing or inhibiting cancer cell proliferation in asubject in need thereof, said method comprising administering to saidsubject a therapeutically effective amount of anti-HERV K positiveantibody wherein the cancer cell proliferation is blocked or reduced.10. A method of treating cancer in a subject in need thereof comprisingthe step of administering to said subject a therapeutically effectiveamount of anti-HERV K positive antibody wherein said cancer includesbreast cancer, ovarian cancer, or melanoma.